A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3

A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3 Thea Kristensen, Preben Normann, Graham J. Belsham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5803023/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 6 You are reading this latest preprint version Abstract In 2019, the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) started to spread globally and caused the COVID-19 pandemic. SARS-CoV-2, like other members of the Coronaviridae , has a single-stranded, positive sense RNA genome about 30 kb in length, which is translated to generate 16 non-structural proteins (NSPs); a set of sub-genomic mRNAs encode the structural and accessory proteins. The ORF1a precursor includes NSP1-11 and is processed by virus-encoded proteases to produce the mature proteins. We recently identified a short, highly conserved motif (YCPRP) within the structural protein precursor of foot-and-mouth disease virus (FMDV), a member of the Picornaviridae. This motif is conserved among picornaviruses and is found as (W/F/Y)-x-P-R-(P/A). The motif has a major influence on the processing of the FMDV capsid precursor (P1-2A) by the viral protease 3C pro . We have now identified a similar motif (WVPRA) within the NSP2 of SARS-CoV-2. Interestingly, this motif is required for the efficient processing of the NSP1-NSP2 junction by the SARS-CoV-2 protease PL pro (NSP3) and a single amino acid substitution within the motif can abrogate cleavage of this junction. We hypothesise that this motif acts, within NSP1-NSP2, to enable this precursor to fold correctly and allow efficient processing of the NSP1/NSP2 junction. Biological sciences/Microbiology Biological sciences/Molecular biology SARS-CoV-2 intramolecular chaperone coronavirus NSP2 proteolytic processing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In December 2019, a previously unknown coronavirus caused an outbreak of a respiratory disease among people in the city of Wuhan in China 1 . Since then, the disease has spread globally and caused the COVID-19 pandemic. The causative virus is called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it primarily affects the respiratory system causing influenza-like symptoms such as coughing, fever and in more severe cases, breathing problems 2 . SARS-CoV-2 belongs to the family Coronaviridae , these viruses have a single stranded, positive-sense, RNA genome (26 to 32 kb in length), which is bound by the nucleocapsid (N) protein and enclosed within a lipid envelope that incorporates the other viral structural proteins, namely the spike (S), envelope (E) and membrane (M) proteins. The SARS-CoV-2 RNA genome is capped at its 5´-terminus, polyadenylated at its 3´-terminus and is infectious by itself following entry into cells 3 . The virus family is classified into four genera, alpha-, beta-, gamma-, and delta- coronaviruses 4 , and these four genera are further subdivided into four lineage subgroups (A, B, C and D) 5 . The SARS-CoV-2 (beta-CoV, subgroup B) genome shows a sequence identity of 80.0% to that of SARS-CoV (beta-CoV, subgroup B), 56.9% to the Middle East Respiratory Syndrome (MERS) CoV (beta-CoV, subgroup C), 51.6% to Human coronavirus (HcoV)-HKU1 (beta-CoV, subgroup A) and 50.5% to HcoV-C43 (beta-CoV, subgroup A) 6 . The human alphacoronaviruses HcoV-NL63 and HcoV-229E have a sequence identity of 48.7% and 47.7%, respectively, to the SARS-CoV-2 genome 6 . Coronavirus replication takes place within an extensive membranous network of virus-modified vesicles derived from the endoplasmic reticulum within the cytoplasm 7 . The first 20 kb of the RNA genome, from the 5'-terminus, serves as an mRNA for the synthesis of two large polyproteins from two partially overlapping reading frames termed ORF1a and ORF1b. The latter sequence is only accessed following programmed ribosomal frameshifting 7 . The two polyproteins, termed pp1a and pp1ab, are cleaved by virus-encoded proteases present within the polyproteins, which results in the production of 16 mature non-structural proteins (NSPs), called NSP1 to NSP16 (Fig. 1 ). The proteins encoded by ORF1a are largely involved in the inhibition of the host cellular innate immune responses, whereas the proteins encoded by ORF1b are required for genome replication, e.g. the RNA dependent RNA polymerase. The pp1a includes two viral proteases. These are the Papain-like protease (PL pro or NSP3) and the 3C-like protease (3CL Pro or NSP5). The 3CL Pro is responsible for cleavage of the two viral polyproteins at 10 different sites while the PL pro (NSP3) cleaves the pp1a polyprotein at 3 other sites, namely the junctions between NSP1/NSP2, NSP2/NSP3 and NSP3/NSP4 7 , see Fig. 1 . In an earlier study on foot-and-mouth disease virus (FMDV) capsid precursor processing, we identified a highly conserved motif of 5 amino acids (YCPRP), which is present within the VP1 region of the P1-2A capsid precursor 8 of this picornavirus. This motif (with few variations) was also identified in the VP1 of other picornaviruses, including Cardioviruses (which have FCPRP), Hepatoviruses (with YFPRP) and Enteroviruses (with WCPRP). Note, these different forms of this motif each start with an aromatic amino acid (aa) residue (Y, F or W). Single amino acid substitutions within this motif of the FMDV P1-2A resulted in a capsid precursor that was highly resistant to proteolytic cleavage by the FMDV 3C protease (3C pro ) at each of the internal sites 8 , i.e. at the junctions between VP0/VP3 and VP3/VP1, the first of these being more than 400 aa away from the YCPRP motif. This was surprising since, in contrast, we have previously demonstrated that cleavage of these junctions is independent of each other and that blocking cleavage at one site does not prevent cleavage of the other sites 9 , thus indicating the crucial role of this motif in the precursor processing. Furthermore, single substitutions within this conserved motif resulted in the loss of virus infectivity 8 , 10 . Thus, it was suggested that this motif is crucial for the correct folding of the capsid precursor to allow subsequent processing into the mature capsid proteins. We hypothesized that this motif may serve as an important binding site for protein chaperones, which may be involved in achieving the correct folding of the precursor 8 , 10 prior to cleavage. Several studies have reported the involvement of such chaperones, e.g. Hsp70 and Hsp90, in picornavirus capsid processing 11 , 12 . Interestingly, we have now identified a similar, but distinct, motif (WVPRA) in the NSP2 protein of SARS-CoV-2. From sequence alignments, we found that this motif is also present (and thus conserved) in other betacoronaviruses, within subgroup A and B, including SARS-CoV, MERS-CoV, human CoV OC43, bovine CoV, equine CoV and several bat coronaviruses (Fig. 2 ). In this study, we have shown that this motif, within the NSP2 protein of SARS-CoV-2, has high importance for the cleavage of the NSP1-NSP2 junction by PL pro (NSP3) even though it is located far away in the linear sequence. Materials and Methods Plasmid design The SARS-CoV-2 sequence (from Wuhan) was obtained from NCBI (RefSeq: NC_045512.2). The NSP1-NSP2 coding sequence (ca. 2500 nt) was modified by adding the sequence for a C-terminal Flag-tag followed by two stop codons. The entire coding sequence was flanked by restriction sites: BamHI (upstream of the coding sequence) and XhoI, XbaI and ApaI downstream of the Flag-tag. The sequence was inserted into the vector pcDNA3.1 (customer designed and produced by GenScript, USA Inc). Following receipt, the plasmid was transformed into chemically competent Top10 Escherichia coli ( E. coli ) cells, amplified and purified using a Plasmid Midi Kit (Qiagen). The pcDNA3.1 plasmid with its NSP1-NSP-2 cDNA insert and the empty pGEM-3Z vector (Promega) were digested with BamHI and XbaI and the products were separated by gel electrophoresis and purified using a GeneJet Gel Purification Kit (Thermo Fisher Scientific). The insert and the linearized pGEM-3Z vector were joined using T4 DNA ligase (Thermo Fisher Scientific) and transformed into chemically competent E. coli cells, amplified and plated onto LB agar plates containing carbenicillin. Colonies were screened for the presence of the insert by digestion with BamHI and XbaI . Plasmids with the required structure were amplified and purified as described above. Variants of the plasmids were prepared using site directed mutagenesis 13 . Briefly, fragments were amplified using Phusion® High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and the pGEM-3Z containing the wt Flag-tagged NSP1-NSP2 coding sequence as template. For each modification, one primer with the desired mutation was used together with a wt primer and template to generate a megaprimer (120–1200 bp), see Supplementary Table 1. The megaprimers were gel purified using the GeneJet Gel Purification Kit (Thermo Fisher Scientific) and used in a mega PCR together with the template. The mega PCR products were digested with DpnI and transformed into chemically competent E. coli cells. Plasmid DNA from individual colonies was screened by Sanger Sequencing (LGC BiosearchTechnologies, Berlin, Germany) for the correct modifications and positive clones were amplified and purified as above. The plasmid NSP3-mCherry, encoding a functional PL pro , was a gift from Bruno Antonny (Addgene plasmid # 165131 ; http://n2t.net/addgene:165131 ; RRID:Addgene_165131) 14 . The plasmid was modified to contain the T7 promoter sequence upstream of the NSP3 sequence (see Supplementary Table 1) by site-directed mutagenesis as described above. Transient expression assays Baby hamster kidney 21 (BHK-21) cells were seeded into 6-well plates approximately 24 h before starting the transfection assay. The BHK-21 cells were 80–90% confluent when they were infected with the recombinant vaccinia virus, termed vTF7-3, which expresses the T7 RNA polymerase 15 . All the NSP1-NSP2 constructs were in the pGEM-3Z vector, which contains the T7 promoter upstream of the coding sequence. Starting from the NSP3-mCherry plasmid 14 , we modified it to contain the T7 promoter upstream of the NSP3 coding sequence. After a 1 hr incubation with vTF7-3 in 5% CO 2 at 37°C, the medium containing this vaccinia virus was removed and the cells were transfected with the required plasmid DNAs using FuGENE 6 (Promega) as described previously 16 . For each well, 1000 ng of the various NSP1-NSP2 plasmids was used, either alone or in combination with 50 ng of the T7-NSP3-mCherry construct. The cells were incubated in 5% CO 2 at 37°C overnight. The next day, the medium was removed and the cells were lysed with 500 µl Buffer C (20mM Tris-HCl (pH 8.0), 125 mM NaCl and 0.5% NP-40). After harvesting, the cell lysates were clarified by centrifugation at 13,000 x g for 10 minutes at 4°C. Immunoblot analysis Prior to immunoblotting, cell lysates were mixed with an equal volume of 2 x Laemmli sample buffer (Bio-Rad) containing 25 mM dithiothreitol (DTT) and boiled for 5 minutes. The proteins were separated by SDS-PAGE (Any KD Bis-Tris gels (Bio-Rad)) and transferred to a PVDF membrane (Bio-Rad). PBS containing 0.1% Tween (PBST) and 5% bovine serum albumin (BSA) was used as blocking buffer and dilution buffer for both the primary and secondary antibodies. The primary antibody used was anti-Flag rabbit polyclonal antibody (1:1000, Proteintech 20543-1-AP) and the secondary antibody was HRP-conjugated goat anti-rabbit IgG (H + L) (1:500, Thermo Fisher Scientific 32460). Following these incubations, a chemiluminescence kit (Pierce ® ECL Western Blotting Substrate, Thermo Fisher Scientific) was used to detect the target proteins. Images were captured using a Chem-Doc XRS system (Bio-Rad) as described previously 8 . Results Cleavage of the NSP1-NSP2 junction by PL Pro ; the effect of truncating the NSP2 protein on NSP1-NSP2 precursor processing The WVPRA motif identified in NSP2 of SARS-CoV-2 is located 396 aa residues away from the C-terminus of this protein (Fig. 3 ). In contrast, the motif that we identified within the VP1 sequence of FMDV is only 26 residues away its C-terminus. Thus, truncating the FMDV VP1 sequence by removing 26 aa (as performed previously 8 ) seems less drastic than truncating the SARS-CoV-2 NSP2 sequence by 396 residues. In order to test the effect of this motif on the cleavage of the NSP1-NSP2 junction by NSP3, we designed and made different variants of the NSP1-NSP2 construct, terminating at different sites: NSP1-NSP2 (wt, 639 aa in length), NSP1-NSP2 (NSP2 L598Stop), NSP1-NSP2 (G458Stop), NSP1-NSP2 (W243Stop), the latter being the only one that removed the WVPRA motif. Transient expression assays were used to express the NSP1-NSP2 (wt) and the truncated versions, within BHK cells, both in the absence and presence of PL Pro (from the T7-NSP3-mcherry plasmid). The Flag-tagged NSP1-NSP2 (wt) served as a positive control, and yielded the expected, uncleaved, product of approximately 93 kDa when expressed alone (Fig. 4 , lane 1). When the NSP1-NSP2 (wt) was co-expressed with the PL pro , the precursor was efficiently processed to generate NSP1 and NSP2, as indicated by the production of the Flag-tagged-NSP2 at approximately 71 kDa (Fig. 4 , lane 2). The plasmid encoding the mutant precursor NSP1-NSP2 (NSP2 L598Stop) yielded a slightly smaller product (ca. 87 kDa) than the wt (as expected) in the absence of PL pro (Fig. 4 , lane 3) and in the presence of PL pro a shorter cleavage product (ca. 66 kDa) was observed, corresponding to the truncated NSP2 (Fig. 4 , lane 4). Both NSP1-NSP2 (G458Stop) and NSP1-NSP2 (W243Stop) yielded precursor products corresponding to their expected shortened sizes (ca. 71 kDA and 48 kDa, respectively) in the absence of PL pro (Fig. 4 , lane 5 and 7). However, in contrast to the wt protein and the NSP2 L598Stop product (Fig. 4 , lanes 2 and 4), no processing of these truncated proteins was observed in the presence of PL pro (Fig. 4 , lane 6 and 8). Deletion of the WVPRA motif alone prevents processing of the NSP1-NSP2 junction by PL In order to assess the importance of the WVPRA motif within NSP2 for protein processing, a small deletion, removing just the sequence encoding this 5 amino acid motif, was introduced into the NSP1-NSP2 precursor coding sequence, this construct is referred to as NSP1-NSP2 (NSP2 Δ243–247). This construct generated a product very similar in size to the wt protein in the absence of PL pro (Fig. 5 , lane 3). However, in the presence of PL pro , it was observed that this mutant precursor could not be processed (Fig. 5 , lane 4) although this 5 residue deletion was over 240 residues away from the cleavage site. Identification of critical residues within the WVPRA motif for NSP1-NSP2 processing To identify individual residues of high importance within the WVPRA motif, single amino acid substitutions were introduced into it. The wt and the mutant precursors were expressed within cells either in the absence or presence of the PL pro . The NSP1-NSP2 (wt) and each of the mutants all generated the expected, similarly sized, products in the absence of the PL pro (Fig. 6 , odd numbered lanes). In the presence of PL pro , the NSP-1-NSP2 (wt) was cleaved to generate the Flag-tagged NSP2, as above (Fig. 6 a and b, lane 2). However, there was no processing observed for the NSP1-NSP2 (NSP2 W243A) mutant in the presence of PL pro (Fig. 6 a, lane 4). In contrast, processing was observed for both the NSP1-NSP2 (NSP2 W243Y) (Fig. 6 a, lane 6) and NSP1-NSP2 (NSP2 W243F) (Fig. 6 a, lane 8) precursors in which rather conservative amino acid substitutions had been made (W, Y and F are all aromatic residues whereas A, in contrast, is a small non-polar residue). Processing, at a qualitatively similar level as in the wt, could also be observed for the NSP1-NSP2 (R246A) mutant (Fig. 6 b, lane 4) (with a change in a different residue within the motif) in the presence of PL Pro . Cleavage was also observed with the NSP1-NSP2 (P245A) mutant in the presence of PL Pro , however it is noteworthy that for this mutant more unprocessed NSP1-NSP2 precursor remained compared to the wt. The NSP1-NSP2 (P245G) mutant appeared to yield less cleaved NSP2 compared to both the wt and the NSP1-NSP2 (P245A) mutant, suggesting a less efficient cleavage in the presence of PL Pro . Thus, deletion of the WVPRA motif or substitution of the W (to a non-aromatic residue) had a major impact on processing of the NSP1-NSP2 junction, whereas changing the P residue (to G or A) within this motif had a smaller impact on the processing of the NSP1-NSP2 junction. Discussion The function of the NSP2 in coronaviruses is not well defined. It has been proposed that the SARS-CoV NSP2 is involved in disrupting intracellular host signaling through interaction with two host proteins termed prohibitin 1 and 2 17 . The NSP2 coding sequence has been completely deleted from the RNA genome of Murine Hepatitis Virus (MHV) and SARS-CoV, and these mutant viruses could still be rescued, proving that NSP2 is not essential for virus growth in cell culture 18 . However, these viruses showed attenuated viral growth and RNA synthesis compared to the wt 18 . One study has suggested that the SARS-CoV-2 NSP2 stimulates translation. In HEK293T-cells, expression of the NSP2 protein increased the protein synthesis rate from mRNAs using both cap- and IRES-dependent translation initiation mechanisms 19 . Another study showed that SARS-CoV-2 NSP2 impedes translation of Ifnb1 transcripts, which encode type I interferon β (IFN-β), by interacting with the GIGYF2 protein 20 . This interaction increases the binding of the GIGYF2 protein to the mRNA cap-binding protein 4EHP, thereby repressing translation of the Ifnb mRNA. Furthermore, analysis of the NSP2 structure, obtained using cryo-electron microscopy and structure prediction, revealed a highly conserved zinc ion-binding site, as present in a number of RNA binding proteins such as RNA polymerases and ribosomes, suggesting a possible role for NSP2 in such interactions 21 . We recently identified a short motif (YCPRP) within the FMDV P1-2A precursor (VP1 in the mature capsid protein) and found it to be conserved among all FMDVs. Furthermore, this motif was also highly conserved across many members of the Picornavirus family, including within cardioviruses, hepatoviruses and enteroviruses. When the motif was modified in the context of FMDV, processing of the FMDV P1-2A precursor by the 3C pro was blocked 8 . This was consistent with earlier studies by Ryan et al., 22 indicating that truncation of the P1-2A capsid precursor blocked processing in vitro . Furthermore, another earlier study had observed a similar effect with the poliovirus capsid precursor (P1) processing by 3CD pro , when the C-terminus of P1 (including the closely related WCPRP motif) was removed then processing of the residual P1 was blocked 23 . On the basis of the results from both SARS-CoV-2 (as described here) and FMDV 8 , 10 , it seems likely that the overall structures of the precursor proteins are adversely affected upon changing or deleting amino acids within these distinct, but closely related, motifs. This would explain why the proteases are not able to recognize the unchanged cleavage sites even when these are located far from the motif in the linear sequence. The short conserved motif (YCPRP) within the C-terminus of FMDV VP1 is extremely important for the cleavage of the capsid precursor P1-2A by 3C pro 8 . Deleting this motif within the FMDV capsid precursor completely abrogated its processing even though the cleavage sites at the VP0/VP3 and VP3/VP1 junctions were located more than 400 aa and around 190 aa away from the deleted motif, respectively. Here, we have now shown that the related motif (WVPRA) within NSP2 of SARS-CoV-2 is necessary for efficient processing of the NSP1/NSP2 junction by the PL pro . Deletion of the WVPRA motif completely blocked processing of the NSP1-NSP2 junction by PL pro (see Fig. 5 , lane 4). We have also analyzed more subtle changes in these motifs. The substitution of the first aromatic amino acids in these motifs, Y in the FMDV P1-2A precursor and the W in the SARS-CoV-2 NSP1-NSP2 precursor, to an A had similarly adverse effects on cleavage of the two precursors, indeed no cleavage was observed for these two mutants. Substitution of the second amino acid in the motif, C to an A, in the FMDV P1-2A precursor, did not have any obvious effect on cleavage, it is also noteworthy that this amino acid is rather non-conserved among different picornaviruses. However, surprisingly, substitution of the third residue P to an A in the FMDV P1-2A precursor, also did not have any apparent effect on the cleavage assays although this amino acid is fully conserved amongst all the picornaviruses that we checked including various cardioviruses, hepatoviruses and enteroviruses 8 . Furthermore, a mutant FMDV RNA transcript encoding the VP1 P187A substitution did not produce infectious virus following introduction into cells, clearly indicating the importance of this residue. Due to these observations with FMDV and the fact that the residue is conserved in beta-coronaviruses, we also tested the effect on cleavage of changing proline to an alanine in the WVPRA motif. Cleavage was observed for the SARS-CoV-2 NSP1-NSP2 (NSP2 P245A) mutant, however it seemed that the cleavage efficiency was less efficient when compared to the wt, as there was still residual precursor detected (Fig. 6 b, lane 6). To investigate the effect of this residue further, the proline in the WVPRA motif was also changed to a glycine. Less cleavage of the NSP1-NSP2 junction was observed for this mutant (Fig. 6 b, lane 8) compared to the alanine substitution (Fig. 6 b, lane 6), suggesting that the alanine residue is tolerated better. Glycine and proline residues within proteins have unusual properties; proline (as an imino acid) introduces a “kink” into the polypeptide chain and glycine residues allow a much greater degree of chain conformations than other residues. Hence, the proline to glycine substitution had been expected to be less detrimental to the protein structure than the alanine substitution but it appeared that the effect of the P to G substitution on cleavage was greater than the P to A change. The FMDV VP1 R188 residue had a huge effect on cleavage of the capsid precursor by the 3C pro 8 . Furthermore, the FMDV (VP1 R188A) substitution also adversely affected the efficiency of virus rescue from RNA and a secondary compensatory substitution, W129R in VP2, was observed after a few passages of the recovered virus in cells 10 . Interestingly, we found that introducing this secondary VP2 (W129R) substitution in the FMDV P1-2A (VP1 R188A) mutant could reverse the block on cleavage, thus showing the importance of the interaction between amino acids within this conserved motif to other sites in the precursor 10 . The SARS-CoV-2 NSP2 R246 is not as conserved (Fig. 2 ) as the FMDV VP1 R188. However, due to the marked effect observed in FMDV, the importance of this residue in the NSP2 was also tested by substitution to an alanine. The NSP2 R246A substitution did not have a huge effect on the cleavage of the NSP1-NSP2 precursor (Fig. 6 b, lane 4). However, it seemed that the processing had slowed to some degree, since more precursor remained for this mutant, compared to the wt (Fig. 6 b, c.f. lane 4 and lane 2). It is rather hard to compare the truncation of the SARS-CoV-2 NSP1-NSP2 with the truncation of FMDV P1-2A as the motifs are located in quite different positions within the proteins. The SARS-CoV-2 motif is located in the middle of the NSP2 sequence whereas the FMDV motif is located near the C-terminus of the VP1 protein. Thus, truncating the NSP2 sequence to remove the motif by removing 396 residues, involved loss of more than half of the protein. This also means that there is a higher probability of changing the overall structure of the protein. It is noteworthy that removing 41 aa from the C-terminus of NSP2 did not affect cleavage at the NSP1-NSP2 junction. However, removing 181 residues (but retaining the conserved motif) prevented cleavage of the NSP1-NSP2 junction by NSP3, thus suggesting that this drastic truncation did affect the overall structure of the precursor. When 396 aa (inclusive the conserved motif) was removed from the precursor, the NSP1-NSP2 junction was again not cleaved. However, this might result from the effect of the large truncation as well as the effect of deleting the conserved motif that, by itself, is sufficient to block processing. A recent study has investigated the processing by the SARS-CoV-2 PL pro of various small synthetic peptides corresponding to the junctions sequences (P8, P7, P6, P5, P4, P3, P2, P1- P1', P2', P3', P4', P5', P6', P7', P8 ') of NSP1-NSP2- (LMRELNGG-AYTRYVDN), NSP2-NSP3- (NTFTLKGG-APTKVTFG) and NSP3-NSP4 (TKIALKGG-KIVNNWLK) 24 . Surprisingly, the study reported that the PL Pro was not able to process the NSP1-NSP2 junction peptide at all, despite being able to process peptides corresponding to the two other junctions 24 . The NSP3-NSP4 junction was not very efficiently processed, whereas the NSP2-NSP3 junction was very effectively processed, and the authors hypothesized that the proline at residue P2' in the NSP2-NSP3 sequence might influence the effectiveness of the processing. Thus, the P2' residue at the NSP1-NSP2 junction in NSP2 (Y2P ) was changed to a proline in the peptide to investigate the effect on processing 24 . Interestingly the small peptide corresponding to the NSP1-NSP2 junction containing the NSP2 (Y2P) substitution was processed very effectively, clearly indicating the importance of this residue. Furthermore, the P2' residue at the NSP3-NSP4 junction was also changed to a proline NSP4 (I2P) to investigate the effect on this processing. Interestingly this also greatly enhanced processing 24 . It is very likely that there is some kind of interaction, either direct or indirect, between the NSP1-NSP2 junction and the conserved motif which is necessary for correct processing of the junction. The fact that the PL Pro was not able to process the wt NSP1-NSP2 junction peptide 24 further suggests that there are some other regions in the polyprotein that are necessary for correct processing of this specific junction, and it is very easy to propose that the conserved motif is involved in this. The introduction of the P residue may well influence the structure of the short peptide substrate and hence facilitate its recognition, the conserved motif may function in an analogous manner for the whole protein. It is interesting to note that similar motifs exist in various different virus families and within different proteins, i.e. in the structural protein (P1-2A or P1) precursor of different picornaviruses and in the NSP2 of multiple betacoronaviruses. Furthermore, the motif seems to have the same important effect on processing in each case. We have also identified similar motifs (of the form Y/F/W-x-P-R-P/A) in other proteins within other viruses, including: human astrovirus, duck astrovirus, canine astrovirus, bovine astrovirus, porcine astrovirus, macacine betaherpes virus 9, Testudinid alphaherpesvirus 3, human papillomavirus 18, human circovirus, Canis familiaris papillomavirus 13, atypical porcine pestivirus 1 and classical swine fever virus. For some of these viruses, the motif is also located within a precursor protein, which is subsequently processed to generate the mature proteins, as with the SARS-CoV-2 NSP2 and FMDV VP1. However, this motif might also affect folding of proteins that are not part of precursor proteins and this could in turn affect interactions of such proteins with other proteins. Moreover, we also found that a similar motif (WGVRP) is present within the Enterobacteria phage K1F. Interestingly, in Enterobacteria phage K1F, this motif is located within a protein previously defined as an intramolecular chaperone. The intramolecular chaperone is located at the C-terminus of the trimeric tail spike protein and is also referred to as the C-terminal domain (CTD) 25 . This intramolecular chaperone, is part of a larger precursor polyprotein and has been shown to be necessary for correct folding of the precursor polyprotein. After the folding of the precursor, the CTD is cleaved off after a conserved serine residue and this cleavage is necessary to stabilize the native protein complex 25 . Originally, an intramolecular chaperone was defined as a pro-region or a pro-sequence, which is covalently linked to the newly synthesized polypeptide 26 . Here it guides and catalyzes the folding of the protein, but subsequently it is cleaved off and thus does not remain as part of the mature protein. However, it has been suggested by Ma et al., 26 that there is also another type of intermolecular chaperone that is not subsequently removed. These chaperones have been referred to as intramolecular chaperone-like building blocks as they are incorporated into the mature protein 26 . Both groups of intramolecular chaperones play important roles in protein folding and hence in protein function. It is very attractive for viruses to produce proteins that have multiple functions, as the viral coding capacity is quite limited. Thus, it is feasible that a protein that functions as an intramolecular chaperone may also be used within the mature virus as is the case for the FMDV VP1 (where the motif is located). However, the SARS-CoV-2 NSP2 is different from the FMDV VP1, as it is not included in the mature virus particle, and furthermore the functions of this protein are not fully defined. It is noteworthy that both FMDV (plus many other picornaviruses) and SARS-CoV-2 (plus many other betacoronaviruses) contain these similar motifs, and that for FMDV and now SARS-COV-2 it has been shown that modification of these motifs in the polyproteins can have a dramatic effect on cleavage of the polyprotein even though the cleavage sites/site are located far away (in the linear sequence) from the site of the conserved motif. Declarations Author Contribution: TK and GJB were responsible for conceiving the idea, planning and designing this study. TK and PN were responsible for the lab work carried out in this study. TK and GJB analyzed the results. TK wrote the initial draft of the manuscript and GJB read and modified it. Funding: This research was funded by the Danish Veterinary and Food Administration (FVST) as part of the agreement for commissioned work between the Danish Ministry of Food and Agriculture and Fisheries and the University of Copenhagen. Data availability statement: The authors confirm that the data supporting the findings of this study are available within the article and the supplementary materials. The data generated, used and analyzed in this study are available upon request from the corresponding author. Additional Information: The authors declare that they have no competing interest. References Wu, D., Wu, T., Liu, Q. & Yang, Z. The SARS-CoV-2 outbreak: What we know. Int. J. Infect. Dis. 94 , 44–48 (2020). Naqvi, A. A. T. et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. BBA - Mol. Basis Dis. 1866 , 1–17 (2020). Beyerstedt, S., Casaro, E. B. & Rangel, É. B. COVID-19: angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection. Eur. J. Clin. Microbiol. Infect. Dis. 40 , 905–919 (2021). Wu, A. et al. Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China. Cell. Host Microbe . 27 , 325–328 (2020). Murugan, C. et al. COVID-19: A review of newly formed viral clades, pathophysiology, therapeutic strategies and current vaccination tasks. Int. J. Biol. Macromol. 193 , 1165–1200 (2021). Cicaloni, V. et al. A Bioinformatics Approach to Investigate Structural and Non-Structural Proteins in Human Coronaviruses. Front. Genet. 13 , 1–12 (2022). Knoops, K. et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 6 , 1957–1974 (2008). Kristensen, T. & Belsham, G. J. Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites. PLoS Pathog 15 (1), (2019). Kristensen, T., Newman, J., Guan, S. H., Tuthill, T. J. & Belsham, G. J. Cleavages at the three junctions within the foot-and-mouth disease virus capsid precursor (P1–2A) by the 3C protease are mutually independent. Virology 522 , 260–270 (2018). Kristensen, T. & Belsham, G. J. Identification of plasticity and interactions of a highly conserved motif within a picornavirus capsid precursor required for virus infectivity. Sci. Rep. 9 , 1–10 (2019). Macejak, D. G. & Sarnow, P. Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. J. Virol. 66 , 1520–1527 (1992). Newman, J. et al. The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers. J. Virol. 92 , 1–14 (2018). Chen, G. J., Qiu, N., Karrer, C., Caspers, P. & Page, M. G. P. Restriction site-free insertion of PCR products directionally into vectors. Biotechniques 28 , 498–505 (2000). Miserey-Lenkei, S. et al. A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types. Biol. Cell. 113 , 311–328 (2021). Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 83, 8122–8126 (1986). Belsham, G. J., Nielsen, I., Normann, P., Royall, E. & Roberts, L. O. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism. RNA 14 , 1671–1680 (2008). Cornillez-Ty, C. T., Liao, L., Yates, J. R., Kuhn, P. & Buchmeier, M. J. Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling. J. Virol. 83 , 10314–10318 (2009). Graham, R. L., Sims, A. C., Baric, R. S. & Denison, M. R. The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication. Adv. Exp. Med. Biol. 581 , 67–72 (2006). Korneeva, N. et al. SARS–CoV–2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions. Virol. J. 1–20. 10.1186/s12985-023-02021-2 (2023). Xu, Z. et al. SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation. Proc. Natl. Acad. Sci. U. S. A. 119, 1–9 (2022). Gupta, M. et al. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. bioRxiv [Preprint]. (2021). 10.1101/2021.05.10.443524 (2021). Ryan, M. D., Belsham, G. J. & King, A. M. Q. Specificity of enzyme-substrate interactions in foot-and-mouth disease virus polyprotein processing. Virology 173 , 35–45 (1989). Ypma-Wong, M. F. & Semler, B. L. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. J. Virol. 61 , 3181–3189 (1987). Chan, H. T. H. et al. Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2′ proline of the viral polyprotein. RSC Chem. Biol. 5 , 117–130 (2023). Schwarzer, D., Stummeyer, K., Gerardy-Schahn, R. & Mühlenhoff, M. Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins. J. Biol. Chem. 282 , 2821–2831 (2007). Ma, B., Tsai, C. J. & Nussinov, R. Binding and folding: In search of intramolecular chaperone-like building block fragments. Protein Eng. 13 , 617–627 (2000). Additional Declarations No competing interests reported. Supplementary Files Supplemetaryfiles16.01.2025Final.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 May, 2025 Reviews received at journal 06 May, 2025 Reviewers agreed at journal 14 Apr, 2025 Reviewers invited by journal 14 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 04 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5803023","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":442940785,"identity":"b9bb3364-5924-490a-b7ee-4a579cb19f11","order_by":0,"name":"Thea Kristensen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2PsWrDMBCGL4uyXJNVxiF6BQWDCWTIqygY4sVuhy4dMhgM6St4yyv4ERwEmlS6aiiFUsjUwVAo7lIqE9IlNsnYQd9woP/40H8ADsd/hLajAjy9p8ewslN3G/inkGMQXK3ASVlll5Sln+9r0K8TtsvVW/PwEu+KdG9ASxjpqvuXiYoomHvkisQB6kNamttoDkaC95T1FEs4hVogJxj6g61MS5qENpHAn/tuuftsWoVtx1/e94+MWXFRSYgtJhAUEnqTSQGmVWwx3lfMrMO50LaYWoc+Kjkr9Udgkxi9nvOHRfRuaiWWLJcHr9lIxh6TmU0W05EW3c1azlc2wbPQ4XA4HFfzC4YbYPD9J8l6AAAAAElFTkSuQmCC","orcid":"","institution":"University of Copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Thea","middleName":"","lastName":"Kristensen","suffix":""},{"id":442940786,"identity":"84cadf3a-e3f5-4241-b791-273206276fd8","order_by":1,"name":"Preben Normann","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Preben","middleName":"","lastName":"Normann","suffix":""},{"id":442940787,"identity":"e0b1894b-1717-4c89-bb5a-a1a11be6c7d6","order_by":2,"name":"Graham J. Belsham","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Graham","middleName":"J.","lastName":"Belsham","suffix":""}],"badges":[],"createdAt":"2025-01-10 10:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5803023/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5803023/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-10244-2","type":"published","date":"2025-07-16T16:05:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80757227,"identity":"55259fc8-8aca-47ac-afbc-b137ed33e1d4","added_by":"auto","created_at":"2025-04-16 18:09:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":503133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the SARS-CoV-2 genome organization.\u003c/strong\u003e The RNA genome is translated from two overlapping reading frames, ORF1a and ORF1b, to generate two different polyproteins, pp1a and pp1ab, the latter is generated following a programmed ribosomal frameshift that occurs within the overlap between ORF1a and ORF1b. The two polyproteins are cleaved by the virus-encoded proteases PL\u003csup\u003epro\u003c/sup\u003e (NSP3) and 3CL\u003csup\u003epro\u003c/sup\u003e (NSP5) into a total of 16 non-structural proteins (NSPs). The sites cleaved by the PL\u003csup\u003epro\u003c/sup\u003e (marked with red arrows) and 3CL\u003csup\u003epro\u003c/sup\u003e (marked with yellow arrows) are indicated. Downstream of the ORF1b, the RNA genome encodes 10 more proteins, which are translated from sub-genomic mRNAs, including the structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) and several accessory proteins. The conserved motif (WVPRA) is found within NSP2 of pp1a and is marked on the lower part of the figure. Created in BioRender. Kristensen, T. (2025) https://BioRender.com/s80a354\u003c/p\u003e","description":"","filename":"Fig1Final.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/7669b9a7ece8c6e65483beea.jpeg"},{"id":80757565,"identity":"c42875b1-c441-4a7f-a52d-04a1fed01841","added_by":"auto","created_at":"2025-04-16 18:17:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment of partial NSP2 amino acid sequences containing the conserved motif from 9 different betacoronaviruses.\u003c/strong\u003e The alignment was performed using ClustalW in Geneious 9.0.5. The entire ORF1a was included in the alignment but just a short region of the NSP2 sequence is shown here. The amino acids are numbered based on the sequence of the SARS-CoV-2 NSP2. The conserved motif is highlighted in different colors depending on their amino acid properties, i.e. W and F have similar properties (aromatic and non-polar) and thus have a similar color. V, L, I and A have similar properties to each other (aliphatic and non-polar) so also have a similar color. Only the P residue (which has distinct properties as an imino acid) is totally conserved. The R-V/I change is rather non-conservative. A consensus sequence from the alignment is shown in the bottom of the Figure where x represents any amino acid. The sequences used were as follows: SARS-CoV-2 (Beta/subB) (GenBank accession number QWC81719.1), SARS-CoV (Beta/subB) (RefSeq accession number YP_009944365.1), Bat coronavirus 279/2005(Beta/SubB) (GenBank accession number GCA_031121315.1), Bat SARS coronavirus HKU3(Beta/SubB) (GenBank accession number QND76018.1), MERS-CoV (Beta/SubB) (GenBank accession number AVN89452.1), Murine hepatitis virus(Beta/subA) (GenBank accession number AWB14623.1), Human coronavirus OC43(Beta/subA) (GenBank accession number GCA_003972325.1), Equine coronavirus(Beta/subA) (GenBank accession number UGN73921.1) and Bovine coronavirus(Beta/subA) (GenBank accession number UZT75406.1).\u003c/p\u003e","description":"","filename":"Fig2Final.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/84bd0fe4eb6177efa660a534.jpg"},{"id":80758050,"identity":"c11411ba-5da7-4eac-a1ef-07a148dae9e3","added_by":"auto","created_at":"2025-04-16 18:25:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":504633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the coding capacity of plasmids used in the transient expression assays. a) \u003c/strong\u003eThe coding sequence for the SARS-CoV-2 NSP1-NSP2 plus a C-terminal Flag-tag and a stop codon was inserted into the pGEM-3Z plasmid, containing the T7 promoter upstream of the insert. The lengths of both NSP1 and NSP2 (in terms of the number of amino acid residues) are indicated, and the location of the conserved motif (WVPRA) is marked. This plasmid is referred to as the wt. \u003cstrong\u003eb)\u003c/strong\u003e The coding sequence for NSP2 was truncated to terminate after aa 598, thus removing 41 aa. \u003cstrong\u003ec) \u003c/strong\u003eThe coding sequence for NSP2 was truncated to terminate after aa 458, thus removing 181 aa. \u003cstrong\u003ed) \u003c/strong\u003eThe coding sequence for NSP2 was truncated to terminate after aa 243, thus removing 396 aa (including the WVPRA motif). In each case, the Flag-tag was linked directly to the C-terminus of the NSP2 sequences within a continuous reading frame so that a NSP1-NSP2-Flag fusion protein was encoded. \u003cstrong\u003ee)\u003c/strong\u003e The NSP3-mCherry construct, encoding a functional PL\u003csup\u003epro\u003c/sup\u003e, \u0026nbsp;was a gift from Bruno Antonny (see Materials and Methods) \u003csup\u003e14\u003c/sup\u003e, a T7 promoter sequence was inserted into this plasmid, upstream of the coding sequence for NSP3, as described in Materials \u0026amp; Methods. Created in BioRender. Kristensen, T. (2025) https://BioRender.com/p25s056\u003c/p\u003e","description":"","filename":"Fig3Final.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/d211720737d246aaa5a6d51b.jpeg"},{"id":80757566,"identity":"22705700-62cb-4c1a-b77a-72705e84278c","added_by":"auto","created_at":"2025-04-16 18:17:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the processing of the SARS-CoV-2 NSP1/NSP2 junction by PL\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003epro\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e after truncations of the NSP2.\u003c/strong\u003e Cell lysates from BHK cells (infected with vTF7-3 \u003csup\u003e15\u003c/sup\u003e) that had been transfected with plasmids (see Figure 3) that express the wt or mutant forms of the SARS-CoV-2 NSP1-NSP2-Flag fusion proteins, either alone (odd numbered lanes) or with the plasmid encoding the SARS-CoV-2 PL\u003csup\u003epro\u003c/sup\u003e (even numbered lanes) were analyzed by immunoblotting using rabbit anti-Flag antibodies, followed by anti-rabbit HRP-conjugated secondary antibodies and a chemiluminescence detection kit. The migration of molecular mass markers (kDa) is indicated on the left. A negative control (no DNA) was included (lane 9). The uncleaved NSP1-NSP2-Flag products are marked with blue arrows and the NSP2-Flag cleavage products are marked with red arrows. The uncut membrane is shown in Supplementary Fig.S1. This experiment has been repeated with very similar results and the key observations have also been obtained using a different plasmid to express the PL\u003csup\u003epro\u003c/sup\u003e and yielded similar results also. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig4Final.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/9d7e2a4e0b41ed081af552d5.jpg"},{"id":80757230,"identity":"8af95e7a-f476-4851-a25e-4e1bd466fa8f","added_by":"auto","created_at":"2025-04-16 18:09:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA short, conserved motif within SARS-CoV-2 NSP2 is required for processing of the NSP1/NSP2 junction by the PL\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003epro\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e (NSP3).\u003c/strong\u003e Cell lysates from BHK cells infected with vTF7-3 \u003csup\u003e15\u003c/sup\u003e and transfected with plasmids that express the SARS-CoV-2 NSP1-NSP2-Flag (wt or with a small deletion, removing the conserved motif) either alone (odd numbered lanes) or with the plasmid encoding the SARS-CoV-2 PL\u003csup\u003epro\u003c/sup\u003e (even numbered lanes) were analyzed by immunoblotting as in Figure 4. Molecular mass markers (kDa) are indicated on the left. A negative control (no DNA) was included (lane 5). The uncut membrane is shown in Supplementary Fig.S2. This experiment has been repeated with very similar results and the key observations have also been obtained using a different plasmid to express the PL\u003csup\u003epro\u003c/sup\u003e and yielded similar results also. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig5Final.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/b91d3ab08d90b4fc7c69f298.jpg"},{"id":80757569,"identity":"5e49343b-29d1-46d2-a6f9-65d0522d6ee4","added_by":"auto","created_at":"2025-04-16 18:17:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":475716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of amino acid residues within the conserved WVPRA motif within the SARS-CoV-2 NSP2 that are required for efficient processing of the NSP1/NSP2 junction by PL\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003epro\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e (NSP3). \u003c/strong\u003eCell lysates from BHK cells infected with vTF7-3 \u003csup\u003e15\u003c/sup\u003e and transfected with plasmids that express the SARS-CoV-2 NSP1-NSP2-Flag (wt or with single amino acid substitutions within the conserved WVPRA motif) either alone (odd numbered lanes) or with the plasmid encoding the SARS-CoV-2 PL\u003csup\u003epro\u003c/sup\u003e (even numbered lanes) were analyzed by immunoblotting as in Figure 4. Molecular mass markers (kDa) are indicated on the left. A negative control was included (lane 9). The NSP1-NSP2-Flag and the NSP2-Flag products are indicated on the right of the figure and were detected using anti-Flag-antibodies. Panel a) shows the wt and various substitutions of the NSP2 W243 and a negative control. Panel b) shows the wt, the NSP2 R246A, the NSP2 P247A, NSP2 P247G and a negative control. The uncut membranes are shown in Supplementary Fig. S3 and S4. This experiment has been repeated with very similar results and the key observations have also been obtained using a different plasmid to express the PL\u003csup\u003epro\u003c/sup\u003e and yielded similar results also. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig6Final.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/94b0532bb30aa3307189028f.jpg"},{"id":87220370,"identity":"85903234-b98f-4eef-a1c6-c8550b80b79c","added_by":"auto","created_at":"2025-07-21 16:12:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2951143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/eabe81ff-929b-4704-92a9-f98e2a16592e.pdf"},{"id":80757233,"identity":"348e308a-3e82-4d86-9b5c-9fa73ef653f6","added_by":"auto","created_at":"2025-04-16 18:09:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":226142,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemetaryfiles16.01.2025Final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5803023/v1/c6230e1ffe17c0b784ad7604.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn December 2019, a previously unknown coronavirus caused an outbreak of a respiratory disease among people in the city of Wuhan in China \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Since then, the disease has spread globally and caused the COVID-19 pandemic. The causative virus is called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it primarily affects the respiratory system causing influenza-like symptoms such as coughing, fever and in more severe cases, breathing problems \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 belongs to the family \u003cem\u003eCoronaviridae\u003c/em\u003e, these viruses have a single stranded, positive-sense, RNA genome (26 to 32 kb in length), which is bound by the nucleocapsid (N) protein and enclosed within a lipid envelope that incorporates the other viral structural proteins, namely the spike (S), envelope (E) and membrane (M) proteins. The SARS-CoV-2 RNA genome is capped at its 5\u0026acute;-terminus, polyadenylated at its 3\u0026acute;-terminus and is infectious by itself following entry into cells \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The virus family is classified into four genera, alpha-, beta-, gamma-, and delta- coronaviruses \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and these four genera are further subdivided into four lineage subgroups (A, B, C and D) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The SARS-CoV-2 (beta-CoV, subgroup B) genome shows a sequence identity of 80.0% to that of SARS-CoV (beta-CoV, subgroup B), 56.9% to the Middle East Respiratory Syndrome (MERS) CoV (beta-CoV, subgroup C), 51.6% to Human coronavirus (HcoV)-HKU1 (beta-CoV, subgroup A) and 50.5% to HcoV-C43 (beta-CoV, subgroup A) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The human alphacoronaviruses HcoV-NL63 and HcoV-229E have a sequence identity of 48.7% and 47.7%, respectively, to the SARS-CoV-2 genome \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCoronavirus replication takes place within an extensive membranous network of virus-modified vesicles derived from the endoplasmic reticulum within the cytoplasm \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The first 20 kb of the RNA genome, from the 5'-terminus, serves as an mRNA for the synthesis of two large polyproteins from two partially overlapping reading frames termed ORF1a and ORF1b. The latter sequence is only accessed following programmed ribosomal frameshifting \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The two polyproteins, termed pp1a and pp1ab, are cleaved by virus-encoded proteases present within the polyproteins, which results in the production of 16 mature non-structural proteins (NSPs), called NSP1 to NSP16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The proteins encoded by ORF1a are largely involved in the inhibition of the host cellular innate immune responses, whereas the proteins encoded by ORF1b are required for genome replication, e.g. the RNA dependent RNA polymerase. The pp1a includes two viral proteases. These are the Papain-like protease (PL\u003csup\u003epro\u003c/sup\u003e or NSP3) and the 3C-like protease (3CL\u003csup\u003ePro\u003c/sup\u003e or NSP5). The 3CL\u003csup\u003ePro\u003c/sup\u003e is responsible for cleavage of the two viral polyproteins at 10 different sites while the PL\u003csup\u003epro\u003c/sup\u003e (NSP3) cleaves the pp1a polyprotein at 3 other sites, namely the junctions between NSP1/NSP2, NSP2/NSP3 and NSP3/NSP4 \u003csup\u003e7\u003c/sup\u003e, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn an earlier study on foot-and-mouth disease virus (FMDV) capsid precursor processing, we identified a highly conserved motif of 5 amino acids (YCPRP), which is present within the VP1 region of the P1-2A capsid precursor \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e of this picornavirus. This motif (with few variations) was also identified in the VP1 of other picornaviruses, including \u003cem\u003eCardioviruses\u003c/em\u003e (which have FCPRP), \u003cem\u003eHepatoviruses\u003c/em\u003e (with YFPRP) and \u003cem\u003eEnteroviruses\u003c/em\u003e (with WCPRP). Note, these different forms of this motif each start with an aromatic amino acid (aa) residue (Y, F or W). Single amino acid substitutions within this motif of the FMDV P1-2A resulted in a capsid precursor that was highly resistant to proteolytic cleavage by the FMDV 3C protease (3C\u003csup\u003epro\u003c/sup\u003e) at each of the internal sites \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, i.e. at the junctions between VP0/VP3 and VP3/VP1, the first of these being more than 400 aa away from the YCPRP motif. This was surprising since, in contrast, we have previously demonstrated that cleavage of these junctions is independent of each other and that blocking cleavage at one site does not prevent cleavage of the other sites \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, thus indicating the crucial role of this motif in the precursor processing. Furthermore, single substitutions within this conserved motif resulted in the loss of virus infectivity \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thus, it was suggested that this motif is crucial for the correct folding of the capsid precursor to allow subsequent processing into the mature capsid proteins. We hypothesized that this motif may serve as an important binding site for protein chaperones, which may be involved in achieving the correct folding of the precursor \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e prior to cleavage. Several studies have reported the involvement of such chaperones, e.g. Hsp70 and Hsp90, in picornavirus capsid processing \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, we have now identified a similar, but distinct, motif (WVPRA) in the NSP2 protein of SARS-CoV-2. From sequence alignments, we found that this motif is also present (and thus conserved) in other betacoronaviruses, within subgroup A and B, including SARS-CoV, MERS-CoV, human CoV OC43, bovine CoV, equine CoV and several bat coronaviruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In this study, we have shown that this motif, within the NSP2 protein of SARS-CoV-2, has high importance for the cleavage of the NSP1-NSP2 junction by PL\u003csub\u003epro\u003c/sub\u003e (NSP3) even though it is located far away in the linear sequence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid design\u003c/h2\u003e \u003cp\u003eThe SARS-CoV-2 sequence (from Wuhan) was obtained from NCBI (RefSeq: NC_045512.2). The NSP1-NSP2 coding sequence (ca. 2500 nt) was modified by adding the sequence for a C-terminal Flag-tag followed by two stop codons. The entire coding sequence was flanked by restriction sites: \u003cem\u003eBamHI\u003c/em\u003e (upstream of the coding sequence) and \u003cem\u003eXhoI, XbaI\u003c/em\u003e and \u003cem\u003eApaI\u003c/em\u003e downstream of the Flag-tag. The sequence was inserted into the vector pcDNA3.1 (customer designed and produced by GenScript, USA Inc). Following receipt, the plasmid was transformed into chemically competent Top10 \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) cells, amplified and purified using a Plasmid Midi Kit (Qiagen).\u003c/p\u003e \u003cp\u003eThe pcDNA3.1 plasmid with its NSP1-NSP-2 cDNA insert and the empty pGEM-3Z vector (Promega) were digested with \u003cem\u003eBamHI\u003c/em\u003e and \u003cem\u003eXbaI\u003c/em\u003e and the products were separated by gel electrophoresis and purified using a GeneJet Gel Purification Kit (Thermo Fisher Scientific). The insert and the linearized pGEM-3Z vector were joined using T4 DNA ligase (Thermo Fisher Scientific) and transformed into chemically competent \u003cem\u003eE. coli\u003c/em\u003e cells, amplified and plated onto LB agar plates containing carbenicillin. Colonies were screened for the presence of the insert by digestion with \u003cem\u003eBamHI\u003c/em\u003e and \u003cem\u003eXbaI\u003c/em\u003e. Plasmids with the required structure were amplified and purified as described above.\u003c/p\u003e \u003cp\u003eVariants of the plasmids were prepared using site directed mutagenesis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Briefly, fragments were amplified using Phusion\u0026reg; High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and the pGEM-3Z containing the wt Flag-tagged NSP1-NSP2 coding sequence as template. For each modification, one primer with the desired mutation was used together with a wt primer and template to generate a megaprimer (120\u0026ndash;1200 bp), see Supplementary Table\u0026nbsp;1. The megaprimers were gel purified using the GeneJet Gel Purification Kit (Thermo Fisher Scientific) and used in a mega PCR together with the template. The mega PCR products were digested with \u003cem\u003eDpnI\u003c/em\u003e and transformed into chemically competent \u003cem\u003eE. coli\u003c/em\u003e cells. Plasmid DNA from individual colonies was screened by Sanger Sequencing (LGC BiosearchTechnologies, Berlin, Germany) for the correct modifications and positive clones were amplified and purified as above.\u003c/p\u003e \u003cp\u003eThe plasmid NSP3-mCherry, encoding a functional PL\u003csup\u003epro\u003c/sup\u003e, was a gift from Bruno Antonny (Addgene plasmid # 165131 ; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:165131\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:165131\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ; RRID:Addgene_165131) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The plasmid was modified to contain the T7 promoter sequence upstream of the NSP3 sequence (see Supplementary Table\u0026nbsp;1) by site-directed mutagenesis as described above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransient expression assays\u003c/h3\u003e\n\u003cp\u003eBaby hamster kidney 21 (BHK-21) cells were seeded into 6-well plates approximately 24 h before starting the transfection assay. The BHK-21 cells were 80\u0026ndash;90% confluent when they were infected with the recombinant vaccinia virus, termed vTF7-3, which expresses the T7 RNA polymerase \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. All the NSP1-NSP2 constructs were in the pGEM-3Z vector, which contains the T7 promoter upstream of the coding sequence. Starting from the NSP3-mCherry plasmid \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we modified it to contain the T7 promoter upstream of the NSP3 coding sequence. After a 1 hr incubation with vTF7-3 in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C, the medium containing this vaccinia virus was removed and the cells were transfected with the required plasmid DNAs using FuGENE 6 (Promega) as described previously \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For each well, 1000 ng of the various NSP1-NSP2 plasmids was used, either alone or in combination with 50 ng of the T7-NSP3-mCherry construct. The cells were incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C overnight. The next day, the medium was removed and the cells were lysed with 500 \u0026micro;l Buffer C (20mM Tris-HCl (pH 8.0), 125 mM NaCl and 0.5% NP-40). After harvesting, the cell lysates were clarified by centrifugation at 13,000 x g for 10 minutes at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eImmunoblot analysis\u003c/h3\u003e\n\u003cp\u003ePrior to immunoblotting, cell lysates were mixed with an equal volume of 2 x Laemmli sample buffer (Bio-Rad) containing 25 mM dithiothreitol (DTT) and boiled for 5 minutes. The proteins were separated by SDS-PAGE (Any KD Bis-Tris gels (Bio-Rad)) and transferred to a PVDF membrane (Bio-Rad). PBS containing 0.1% Tween (PBST) and 5% bovine serum albumin (BSA) was used as blocking buffer and dilution buffer for both the primary and secondary antibodies. The primary antibody used was anti-Flag rabbit polyclonal antibody (1:1000, Proteintech 20543-1-AP) and the secondary antibody was HRP-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (1:500, Thermo Fisher Scientific 32460). Following these incubations, a chemiluminescence kit (Pierce \u0026reg; ECL Western Blotting Substrate, Thermo Fisher Scientific) was used to detect the target proteins. Images were captured using a Chem-Doc XRS system (Bio-Rad) as described previously \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCleavage of the NSP1-NSP2 junction by PL\u003c/b\u003e \u003csup\u003e \u003cb\u003ePro\u003c/b\u003e \u003c/sup\u003e; \u003cb\u003ethe effect of truncating the NSP2 protein on NSP1-NSP2 precursor processing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe WVPRA motif identified in NSP2 of SARS-CoV-2 is located 396 aa residues away from the C-terminus of this protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, the motif that we identified within the VP1 sequence of FMDV is only 26 residues away its C-terminus. Thus, truncating the FMDV VP1 sequence by removing 26 aa (as performed previously \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e) seems less drastic than truncating the SARS-CoV-2 NSP2 sequence by 396 residues. In order to test the effect of this motif on the cleavage of the NSP1-NSP2 junction by NSP3, we designed and made different variants of the NSP1-NSP2 construct, terminating at different sites: NSP1-NSP2 (wt, 639 aa in length), NSP1-NSP2 (NSP2 L598Stop), NSP1-NSP2 (G458Stop), NSP1-NSP2 (W243Stop), the latter being the only one that removed the WVPRA motif. Transient expression assays were used to express the NSP1-NSP2 (wt) and the truncated versions, within BHK cells, both in the absence and presence of PL\u003csup\u003ePro\u003c/sup\u003e (from the T7-NSP3-mcherry plasmid). The Flag-tagged NSP1-NSP2 (wt) served as a positive control, and yielded the expected, uncleaved, product of approximately 93 kDa when expressed alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 1). When the NSP1-NSP2 (wt) was co-expressed with the PL\u003csup\u003epro\u003c/sup\u003e, the precursor was efficiently processed to generate NSP1 and NSP2, as indicated by the production of the Flag-tagged-NSP2 at approximately 71 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 2). The plasmid encoding the mutant precursor NSP1-NSP2 (NSP2 L598Stop) yielded a slightly smaller product (ca. 87 kDa) than the wt (as expected) in the absence of PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 3) and in the presence of PL\u003csup\u003epro\u003c/sup\u003e a shorter cleavage product (ca. 66 kDa) was observed, corresponding to the truncated NSP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 4). Both NSP1-NSP2 (G458Stop) and NSP1-NSP2 (W243Stop) yielded precursor products corresponding to their expected shortened sizes (ca. 71 kDA and 48 kDa, respectively) in the absence of PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 5 and 7). However, in contrast to the wt protein and the NSP2 L598Stop product (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lanes 2 and 4), no processing of these truncated proteins was observed in the presence of PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lane 6 and 8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDeletion of the WVPRA motif alone prevents processing of the NSP1-NSP2 junction by PL\u003c/h3\u003e\n\u003cp\u003eIn order to assess the importance of the WVPRA motif within NSP2 for protein processing, a small deletion, removing just the sequence encoding this 5 amino acid motif, was introduced into the NSP1-NSP2 precursor coding sequence, this construct is referred to as NSP1-NSP2 (NSP2 Δ243\u0026ndash;247). This construct generated a product very similar in size to the wt protein in the absence of PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, lane 3). However, in the presence of PL\u003csup\u003epro\u003c/sup\u003e, it was observed that this mutant precursor could not be processed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, lane 4) although this 5 residue deletion was over 240 residues away from the cleavage site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of critical residues within the WVPRA motif for NSP1-NSP2 processing\u003c/h2\u003e \u003cp\u003eTo identify individual residues of high importance within the WVPRA motif, single amino acid substitutions were introduced into it. The wt and the mutant precursors were expressed within cells either in the absence or presence of the PL\u003csup\u003epro\u003c/sup\u003e. The NSP1-NSP2 (wt) and each of the mutants all generated the expected, similarly sized, products in the absence of the PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, odd numbered lanes). In the presence of PL\u003csup\u003epro\u003c/sup\u003e, the NSP-1-NSP2 (wt) was cleaved to generate the Flag-tagged NSP2, as above (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b, lane 2). However, there was no processing observed for the NSP1-NSP2 (NSP2 W243A) mutant in the presence of PL\u003csup\u003epro\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane 4). In contrast, processing was observed for both the NSP1-NSP2 (NSP2 W243Y) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane 6) and NSP1-NSP2 (NSP2 W243F) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane 8) precursors in which rather conservative amino acid substitutions had been made (W, Y and F are all aromatic residues whereas A, in contrast, is a small non-polar residue). Processing, at a qualitatively similar level as in the wt, could also be observed for the NSP1-NSP2 (R246A) mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, lane 4) (with a change in a different residue within the motif) in the presence of PL\u003csup\u003ePro\u003c/sup\u003e. Cleavage was also observed with the NSP1-NSP2 (P245A) mutant in the presence of PL\u003csup\u003ePro\u003c/sup\u003e, however it is noteworthy that for this mutant more unprocessed NSP1-NSP2 precursor remained compared to the wt. The NSP1-NSP2 (P245G) mutant appeared to yield less cleaved NSP2 compared to both the wt and the NSP1-NSP2 (P245A) mutant, suggesting a less efficient cleavage in the presence of PL\u003csup\u003ePro\u003c/sup\u003e. Thus, deletion of the WVPRA motif or substitution of the W (to a non-aromatic residue) had a major impact on processing of the NSP1-NSP2 junction, whereas changing the P residue (to G or A) within this motif had a smaller impact on the processing of the NSP1-NSP2 junction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe function of the NSP2 in coronaviruses is not well defined. It has been proposed that the SARS-CoV NSP2 is involved in disrupting intracellular host signaling through interaction with two host proteins termed prohibitin 1 and 2 \u003csup\u003e17\u003c/sup\u003e. The NSP2 coding sequence has been completely deleted from the RNA genome of Murine Hepatitis Virus (MHV) and SARS-CoV, and these mutant viruses could still be rescued, proving that NSP2 is not essential for virus growth in cell culture \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, these viruses showed attenuated viral growth and RNA synthesis compared to the wt \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. One study has suggested that the SARS-CoV-2 NSP2 stimulates translation. In HEK293T-cells, expression of the NSP2 protein increased the protein synthesis rate from mRNAs using both cap- and IRES-dependent translation initiation mechanisms \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Another study showed that SARS-CoV-2 NSP2 impedes translation of \u003cem\u003eIfnb1\u003c/em\u003e transcripts, which encode type I interferon β (IFN-β), by interacting with the GIGYF2 protein \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This interaction increases the binding of the GIGYF2 protein to the mRNA cap-binding protein 4EHP, thereby repressing translation of the \u003cem\u003eIfnb\u003c/em\u003e mRNA. Furthermore, analysis of the NSP2 structure, obtained using cryo-electron microscopy and structure prediction, revealed a highly conserved zinc ion-binding site, as present in a number of RNA binding proteins such as RNA polymerases and ribosomes, suggesting a possible role for NSP2 in such interactions \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe recently identified a short motif (YCPRP) within the FMDV P1-2A precursor (VP1 in the mature capsid protein) and found it to be conserved among all FMDVs. Furthermore, this motif was also highly conserved across many members of the \u003cem\u003ePicornavirus\u003c/em\u003e family, including within cardioviruses, hepatoviruses and enteroviruses. When the motif was modified in the context of FMDV, processing of the FMDV P1-2A precursor by the 3C\u003csup\u003epro\u003c/sup\u003e was blocked \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This was consistent with earlier studies by Ryan et al., \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e indicating that truncation of the P1-2A capsid precursor blocked processing \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, another earlier study had observed a similar effect with the poliovirus capsid precursor (P1) processing by 3CD\u003csup\u003epro\u003c/sup\u003e, when the C-terminus of P1 (including the closely related WCPRP motif) was removed then processing of the residual P1 was blocked \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOn the basis of the results from both SARS-CoV-2 (as described here) and FMDV \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, it seems likely that the overall structures of the precursor proteins are adversely affected upon changing or deleting amino acids within these distinct, but closely related, motifs. This would explain why the proteases are not able to recognize the unchanged cleavage sites even when these are located far from the motif in the linear sequence. The short conserved motif (YCPRP) within the C-terminus of FMDV VP1 is extremely important for the cleavage of the capsid precursor P1-2A by 3C\u003csup\u003epro \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Deleting this motif within the FMDV capsid precursor completely abrogated its processing even though the cleavage sites at the VP0/VP3 and VP3/VP1 junctions were located more than 400 aa and around 190 aa away from the deleted motif, respectively. Here, we have now shown that the related motif (WVPRA) within NSP2 of SARS-CoV-2 is necessary for efficient processing of the NSP1/NSP2 junction by the PL\u003csup\u003epro\u003c/sup\u003e. Deletion of the WVPRA motif completely blocked processing of the NSP1-NSP2 junction by PL\u003csup\u003epro\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, lane 4). We have also analyzed more subtle changes in these motifs. The substitution of the first aromatic amino acids in these motifs, Y in the FMDV P1-2A precursor and the W in the SARS-CoV-2 NSP1-NSP2 precursor, to an A had similarly adverse effects on cleavage of the two precursors, indeed no cleavage was observed for these two mutants. Substitution of the second amino acid in the motif, C to an A, in the FMDV P1-2A precursor, did not have any obvious effect on cleavage, it is also noteworthy that this amino acid is rather non-conserved among different picornaviruses. However, surprisingly, substitution of the third residue P to an A in the FMDV P1-2A precursor, also did not have any apparent effect on the cleavage assays although this amino acid is fully conserved amongst all the picornaviruses that we checked including various cardioviruses, hepatoviruses and enteroviruses \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, a mutant FMDV RNA transcript encoding the VP1 P187A substitution did not produce infectious virus following introduction into cells, clearly indicating the importance of this residue. Due to these observations with FMDV and the fact that the residue is conserved in beta-coronaviruses, we also tested the effect on cleavage of changing proline to an alanine in the WVPRA motif. Cleavage was observed for the SARS-CoV-2 NSP1-NSP2 (NSP2 P245A) mutant, however it seemed that the cleavage efficiency was less efficient when compared to the wt, as there was still residual precursor detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, lane 6). To investigate the effect of this residue further, the proline in the WVPRA motif was also changed to a glycine. Less cleavage of the NSP1-NSP2 junction was observed for this mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, lane 8) compared to the alanine substitution (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, lane 6), suggesting that the alanine residue is tolerated better. Glycine and proline residues within proteins have unusual properties; proline (as an imino acid) introduces a \u0026ldquo;kink\u0026rdquo; into the polypeptide chain and glycine residues allow a much greater degree of chain conformations than other residues. Hence, the proline to glycine substitution had been expected to be less detrimental to the protein structure than the alanine substitution but it appeared that the effect of the P to G substitution on cleavage was greater than the P to A change. The FMDV VP1 R188 residue had a huge effect on cleavage of the capsid precursor by the 3C\u003csup\u003epro \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, the FMDV (VP1 R188A) substitution also adversely affected the efficiency of virus rescue from RNA and a secondary compensatory substitution, W129R in VP2, was observed after a few passages of the recovered virus in cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that introducing this secondary VP2 (W129R) substitution in the FMDV P1-2A (VP1 R188A) mutant could reverse the block on cleavage, thus showing the importance of the interaction between amino acids within this conserved motif to other sites in the precursor \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The SARS-CoV-2 NSP2 R246 is not as conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) as the FMDV VP1 R188. However, due to the marked effect observed in FMDV, the importance of this residue in the NSP2 was also tested by substitution to an alanine. The NSP2 R246A substitution did not have a huge effect on the cleavage of the NSP1-NSP2 precursor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, lane 4). However, it seemed that the processing had slowed to some degree, since more precursor remained for this mutant, compared to the wt (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c.f. lane 4 and lane 2).\u003c/p\u003e \u003cp\u003eIt is rather hard to compare the truncation of the SARS-CoV-2 NSP1-NSP2 with the truncation of FMDV P1-2A as the motifs are located in quite different positions within the proteins. The SARS-CoV-2 motif is located in the middle of the NSP2 sequence whereas the FMDV motif is located near the C-terminus of the VP1 protein. Thus, truncating the NSP2 sequence to remove the motif by removing 396 residues, involved loss of more than half of the protein. This also means that there is a higher probability of changing the overall structure of the protein. It is noteworthy that removing 41 aa from the C-terminus of NSP2 did not affect cleavage at the NSP1-NSP2 junction. However, removing 181 residues (but retaining the conserved motif) prevented cleavage of the NSP1-NSP2 junction by NSP3, thus suggesting that this drastic truncation did affect the overall structure of the precursor. When 396 aa (inclusive the conserved motif) was removed from the precursor, the NSP1-NSP2 junction was again not cleaved. However, this might result from the effect of the large truncation as well as the effect of deleting the conserved motif that, by itself, is sufficient to block processing.\u003c/p\u003e \u003cp\u003eA recent study has investigated the processing by the SARS-CoV-2 PL\u003csup\u003epro\u003c/sup\u003e of various small synthetic peptides corresponding to the junctions sequences (P8, P7, P6, P5, P4, P3, P2, P1- P1', P2', P3', P4', P5', P6', P7', P8 ') of NSP1-NSP2- (LMRELNGG-AYTRYVDN), NSP2-NSP3- (NTFTLKGG-APTKVTFG) and NSP3-NSP4 (TKIALKGG-KIVNNWLK) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Surprisingly, the study reported that the PL\u003csup\u003ePro\u003c/sup\u003e was not able to process the NSP1-NSP2 junction peptide at all, despite being able to process peptides corresponding to the two other junctions\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The NSP3-NSP4 junction was not very efficiently processed, whereas the NSP2-NSP3 junction was very effectively processed, and the authors hypothesized that the proline at residue P2' in the NSP2-NSP3 sequence might influence the effectiveness of the processing. Thus, the P2' residue at the NSP1-NSP2 junction in NSP2 (Y2P ) was changed to a proline in the peptide to investigate the effect on processing \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Interestingly the small peptide corresponding to the NSP1-NSP2 junction containing the NSP2 (Y2P) substitution was processed very effectively, clearly indicating the importance of this residue. Furthermore, the P2' residue at the NSP3-NSP4 junction was also changed to a proline NSP4 (I2P) to investigate the effect on this processing. Interestingly this also greatly enhanced processing \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is very likely that there is some kind of interaction, either direct or indirect, between the NSP1-NSP2 junction and the conserved motif which is necessary for correct processing of the junction. The fact that the PL\u003csup\u003ePro\u003c/sup\u003e was not able to process the wt NSP1-NSP2 junction peptide\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e further suggests that there are some other regions in the polyprotein that are necessary for correct processing of this specific junction, and it is very easy to propose that the conserved motif is involved in this. The introduction of the P residue may well influence the structure of the short peptide substrate and hence facilitate its recognition, the conserved motif may function in an analogous manner for the whole protein.\u003c/p\u003e \u003cp\u003eIt is interesting to note that similar motifs exist in various different virus families and within different proteins, i.e. in the structural protein (P1-2A or P1) precursor of different picornaviruses and in the NSP2 of multiple betacoronaviruses. Furthermore, the motif seems to have the same important effect on processing in each case. We have also identified similar motifs (of the form Y/F/W-x-P-R-P/A) in other proteins within other viruses, including: human astrovirus, duck astrovirus, canine astrovirus, bovine astrovirus, porcine astrovirus, macacine betaherpes virus 9, Testudinid alphaherpesvirus 3, human papillomavirus 18, human circovirus, Canis familiaris papillomavirus 13, atypical porcine pestivirus 1 and classical swine fever virus. For some of these viruses, the motif is also located within a precursor protein, which is subsequently processed to generate the mature proteins, as with the SARS-CoV-2 NSP2 and FMDV VP1. However, this motif might also affect folding of proteins that are not part of precursor proteins and this could in turn affect interactions of such proteins with other proteins. Moreover, we also found that a similar motif (WGVRP) is present within the Enterobacteria phage K1F. Interestingly, in Enterobacteria phage K1F, this motif is located within a protein previously defined as an intramolecular chaperone. The intramolecular chaperone is located at the C-terminus of the trimeric tail spike protein and is also referred to as the C-terminal domain (CTD) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This intramolecular chaperone, is part of a larger precursor polyprotein and has been shown to be necessary for correct folding of the precursor polyprotein. After the folding of the precursor, the CTD is cleaved off after a conserved serine residue and this cleavage is necessary to stabilize the native protein complex \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOriginally, an intramolecular chaperone was defined as a pro-region or a pro-sequence, which is covalently linked to the newly synthesized polypeptide \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Here it guides and catalyzes the folding of the protein, but subsequently it is cleaved off and thus does not remain as part of the mature protein. However, it has been suggested by Ma et al.,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e that there is also another type of intermolecular chaperone that is not subsequently removed. These chaperones have been referred to as intramolecular chaperone-like building blocks as they are incorporated into the mature protein \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Both groups of intramolecular chaperones play important roles in protein folding and hence in protein function.\u003c/p\u003e \u003cp\u003eIt is very attractive for viruses to produce proteins that have multiple functions, as the viral coding capacity is quite limited. Thus, it is feasible that a protein that functions as an intramolecular chaperone may also be used within the mature virus as is the case for the FMDV VP1 (where the motif is located). However, the SARS-CoV-2 NSP2 is different from the FMDV VP1, as it is not included in the mature virus particle, and furthermore the functions of this protein are not fully defined. It is noteworthy that both FMDV (plus many other picornaviruses) and SARS-CoV-2 (plus many other betacoronaviruses) contain these similar motifs, and that for FMDV and now SARS-COV-2 it has been shown that modification of these motifs in the polyproteins can have a dramatic effect on cleavage of the polyprotein even though the cleavage sites/site are located far away (in the linear sequence) from the site of the conserved motif.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u003c/strong\u003e TK and GJB were responsible for conceiving the idea, planning and designing this study. TK and PN were responsible for the lab work carried out in this study. TK and GJB analyzed the results. TK wrote the initial draft of the manuscript and GJB read and modified it.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the Danish Veterinary and Food Administration (FVST) as part of the agreement for commissioned work between the Danish Ministry of Food and Agriculture and Fisheries and the University of Copenhagen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e The authors confirm that the data supporting the findings of this study are available within the article and the supplementary materials. The data generated, used and analyzed in this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information:\u003c/strong\u003e The authors declare that they have no competing interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWu, D., Wu, T., Liu, Q. \u0026amp; Yang, Z. The SARS-CoV-2 outbreak: What we know. \u003cem\u003eInt. J. Infect. Dis.\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, 44\u0026ndash;48 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaqvi, A. A. T. et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. \u003cem\u003eBBA - Mol. Basis Dis.\u003c/em\u003e \u003cb\u003e1866\u003c/b\u003e, 1\u0026ndash;17 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeyerstedt, S., Casaro, E. B. \u0026amp; Rangel, \u0026Eacute;. B. COVID-19: angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection. \u003cem\u003eEur. J. Clin. Microbiol. Infect. Dis.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 905\u0026ndash;919 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, A. et al. Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China. \u003cem\u003eCell. Host Microbe\u003c/em\u003e. \u003cb\u003e27\u003c/b\u003e, 325\u0026ndash;328 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurugan, C. et al. COVID-19: A review of newly formed viral clades, pathophysiology, therapeutic strategies and current vaccination tasks. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cb\u003e193\u003c/b\u003e, 1165\u0026ndash;1200 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCicaloni, V. et al. A Bioinformatics Approach to Investigate Structural and Non-Structural Proteins in Human Coronaviruses. \u003cem\u003eFront. Genet.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1\u0026ndash;12 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnoops, K. et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. \u003cem\u003ePLoS Biol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 1957\u0026ndash;1974 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen, T. \u0026amp; Belsham, G. J. Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen, T., Newman, J., Guan, S. H., Tuthill, T. J. \u0026amp; Belsham, G. J. Cleavages at the three junctions within the foot-and-mouth disease virus capsid precursor (P1\u0026ndash;2A) by the 3C protease are mutually independent. \u003cem\u003eVirology\u003c/em\u003e \u003cb\u003e522\u003c/b\u003e, 260\u0026ndash;270 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen, T. \u0026amp; Belsham, G. J. Identification of plasticity and interactions of a highly conserved motif within a picornavirus capsid precursor required for virus infectivity. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 1\u0026ndash;10 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacejak, D. G. \u0026amp; Sarnow, P. Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e, 1520\u0026ndash;1527 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewman, J. et al. The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e, 1\u0026ndash;14 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, G. J., Qiu, N., Karrer, C., Caspers, P. \u0026amp; Page, M. G. P. Restriction site-free insertion of PCR products directionally into vectors. \u003cem\u003eBiotechniques\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 498\u0026ndash;505 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiserey-Lenkei, S. et al. A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types. \u003cem\u003eBiol. Cell.\u003c/em\u003e \u003cb\u003e113\u003c/b\u003e, 311\u0026ndash;328 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuerst, T. R., Niles, E. G., Studier, F. W. \u0026amp; Moss, B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 83, 8122\u0026ndash;8126 (1986).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelsham, G. J., Nielsen, I., Normann, P., Royall, E. \u0026amp; Roberts, L. O. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5\u0026prime; untranslated regions are efficiently translated in cells by a cap-dependent mechanism. \u003cem\u003eRNA\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1671\u0026ndash;1680 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCornillez-Ty, C. T., Liao, L., Yates, J. R., Kuhn, P. \u0026amp; Buchmeier, M. J. Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e, 10314\u0026ndash;10318 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraham, R. L., Sims, A. C., Baric, R. S. \u0026amp; Denison, M. R. The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication. \u003cem\u003eAdv. Exp. Med. Biol.\u003c/em\u003e \u003cb\u003e581\u003c/b\u003e, 67\u0026ndash;72 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorneeva, N. et al. SARS\u0026ndash;CoV\u0026ndash;2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions. \u003cem\u003eVirol. J.\u003c/em\u003e 1\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12985-023-02021-2\u003c/span\u003e\u003cspan address=\"10.1186/s12985-023-02021-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Z. et al. SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 119, 1\u0026ndash;9 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, M. et al. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. bioRxiv [Preprint]. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2021.05.10.443524\u003c/span\u003e\u003cspan address=\"10.1101/2021.05.10.443524\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyan, M. D., Belsham, G. J. \u0026amp; King, A. M. Q. Specificity of enzyme-substrate interactions in foot-and-mouth disease virus polyprotein processing. \u003cem\u003eVirology\u003c/em\u003e \u003cb\u003e173\u003c/b\u003e, 35\u0026ndash;45 (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYpma-Wong, M. F. \u0026amp; Semler, B. L. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 3181\u0026ndash;3189 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan, H. T. H. et al. Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2\u0026prime; proline of the viral polyprotein. \u003cem\u003eRSC Chem. Biol.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 117\u0026ndash;130 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwarzer, D., Stummeyer, K., Gerardy-Schahn, R. \u0026amp; M\u0026uuml;hlenhoff, M. Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e282\u003c/b\u003e, 2821\u0026ndash;2831 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa, B., Tsai, C. J. \u0026amp; Nussinov, R. Binding and folding: In search of intramolecular chaperone-like building block fragments. \u003cem\u003eProtein Eng.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 617\u0026ndash;627 (2000).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, intramolecular chaperone, coronavirus, NSP2, proteolytic processing","lastPublishedDoi":"10.21203/rs.3.rs-5803023/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5803023/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn 2019, the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) started to spread globally and caused the COVID-19 pandemic. SARS-CoV-2, like other members of the \u003cem\u003eCoronaviridae\u003c/em\u003e, has a single-stranded, positive sense RNA genome about 30 kb in length, which is translated to generate 16 non-structural proteins (NSPs); a set of sub-genomic mRNAs encode the structural and accessory proteins. The ORF1a precursor includes NSP1-11 and is processed by virus-encoded proteases to produce the mature proteins.\u003c/p\u003e \u003cp\u003eWe recently identified a short, highly conserved motif (YCPRP) within the structural protein precursor of foot-and-mouth disease virus (FMDV), a member of the \u003cem\u003ePicornaviridae.\u003c/em\u003e This motif is conserved among picornaviruses and is found as (W/F/Y)-x-P-R-(P/A). The motif has a major influence on the processing of the FMDV capsid precursor (P1-2A) by the viral protease 3C\u003csup\u003epro\u003c/sup\u003e. We have now identified a similar motif (WVPRA) within the NSP2 of SARS-CoV-2. Interestingly, this motif is required for the efficient processing of the NSP1-NSP2 junction by the SARS-CoV-2 protease PL\u003csup\u003epro\u003c/sup\u003e (NSP3) and a single amino acid substitution within the motif can abrogate cleavage of this junction. We hypothesise that this motif acts, within NSP1-NSP2, to enable this precursor to fold correctly and allow efficient processing of the NSP1/NSP2 junction.\u003c/p\u003e","manuscriptTitle":"A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 18:08:58","doi":"10.21203/rs.3.rs-5803023/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-12T05:22:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-06T14:46:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301549053169817192728512216520558275456","date":"2025-04-14T23:13:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-14T21:45:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T05:45:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-04T09:02:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"813a8e5d-a7ac-44e0-9e91-2a0d0a83df96","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47156498,"name":"Biological sciences/Microbiology"},{"id":47156499,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-07-21T16:09:46+00:00","versionOfRecord":{"articleIdentity":"rs-5803023","link":"https://doi.org/10.1038/s41598-025-10244-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-16 16:05:44","publishedOnDateReadable":"July 16th, 2025"},"versionCreatedAt":"2025-04-16 18:08:58","video":"","vorDoi":"10.1038/s41598-025-10244-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-10244-2","workflowStages":[]},"version":"v1","identity":"rs-5803023","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5803023","identity":"rs-5803023","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}