The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses

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Schipper , Chiara Aloise , Sereina O. Sutter , Marleen Zwaagstra , Arno L.W. van Vliet , View ORCID Profile Rana Abdelnabi , Bob Ignacio , View ORCID Profile Kimberly M. Bonger , Dagmar Roelofs , Judith M.A. van den Brand , Richard W. Wubbolts , Lucas J.M. Bruurs , View ORCID Profile Hendrik Jan Thibaut , Johan Neyts , Marvin E. Tanenbaum , Frank J.M. van Kuppeveld doi: https://doi.org/10.1101/2025.02.20.639337 Jelle G. Schipper 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chiara Aloise 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sereina O. Sutter 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marleen Zwaagstra 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arno L.W. van Vliet 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rana Abdelnabi 2 KU Leuven, Department of Microbiology , Immunology and Transplantation, Rega Institute, Virology, Antiviral Drug & Vaccine Research Group , KU Leuven, Leuven, Belgium 3 KU Leuven - Department of Microbiology, Immunology and Transplantation , VirusBank Platform, Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rana Abdelnabi Bob Ignacio 4 Department of Synthetic Organic Chemistry, Chemical Biology Lab, Radboud University , Heyendaalseweg 135, 6525AJ Nijmegen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kimberly M. Bonger 4 Department of Synthetic Organic Chemistry, Chemical Biology Lab, Radboud University , Heyendaalseweg 135, 6525AJ Nijmegen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kimberly M. Bonger Dagmar Roelofs 5 Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Judith M.A. van den Brand 5 Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Richard W. Wubbolts 6 Center for Cell Imaging, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lucas J.M. Bruurs 7 Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht , Upssalalaan 8, 3584 CT Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hendrik Jan Thibaut 2 KU Leuven, Department of Microbiology , Immunology and Transplantation, Rega Institute, Virology, Antiviral Drug & Vaccine Research Group , KU Leuven, Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hendrik Jan Thibaut Johan Neyts 2 KU Leuven, Department of Microbiology , Immunology and Transplantation, Rega Institute, Virology, Antiviral Drug & Vaccine Research Group , KU Leuven, Leuven, Belgium 3 KU Leuven - Department of Microbiology, Immunology and Transplantation , VirusBank Platform, Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marvin E. Tanenbaum 7 Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht , Upssalalaan 8, 3584 CT Utrecht, the Netherlands 8 Department of Bionanoscience, Delft University of Technology , 2600 AA Delft, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: F.J.M.vanKuppeveld{at}uu.nl m.tanenbaum{at}hubrecht.eu Frank J.M. van Kuppeveld 1 Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University , Yalelaan 1, 3584 CL Utrecht, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: F.J.M.vanKuppeveld{at}uu.nl m.tanenbaum{at}hubrecht.eu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2A pro and 3C pro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2A pro . Viruses with an inactive 2A pro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2A pro , but not 3C pro , is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-α/β (IFN-α/β) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2A pro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2A pro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication. Introduction Enteroviruses (EVs) are non-enveloped viruses with a single-stranded (ss) RNA genome of positive polarity [(+)ssRNA] that belong to the family Picornaviridae . The genus Enterovirus includes major human pathogens, such as poliovirus (PV), coxsackievirus (CV), echovirus, emerging numbered enteroviruses like EV-A71 and EV-A68, and rhinoviruses (RVs). Overall, there are over 150 serotypes of non-polio enteroviruses and more than 170 rhinovirus serotypes 1 , hence vaccination is not a feasible option for all of them. Consequently, there is an urgent need for broad-spectrum antiviral drugs against enteroviruses, the development of which requires a comprehensive understanding of the enterovirus life cycle. While different enteroviruses use distinct receptors and entry pathways, many post-entry steps are conserved. After accessing the cytosol, the genomic viral RNA (vRNA) is directly translated into a single large polyprotein. As opposed to canonical cap-dependent translation initiation, translation of the viral ORF is initiated via an internal ribosome entry site (IRES). The resulting polyprotein is auto-proteolytically processed by viral proteases (2A pro and 3C pro ) yielding 11 distinct proteins, including the viral RNA-dependent RNA polymerase, viral capsid proteins, and several other viral proteins involved in replication and/or triggering alterations in the host cell environment. After the initial synthesis of viral proteins, vRNA replication starts with the formation of a negative-strand copy of the incoming viral genome to generate a double-stranded (ds)RNA replication intermediate. Notably, cytosolic dsRNA is highly immunogenic, as it can be recognized by cellular sensors that activate antiviral response pathways 2 . The negative-strand copy next serves as a template for synthesis of multiple new (+)ssRNA strands, which either start subsequent rounds of translation and replication or, at late stage of infection, are encapsidated to form new infectious virus particles 3 . To optimize the cellular environment for viral translation and replication, and to suppress cellular antiviral defense mechanisms, viruses have evolved elaborate strategies to perturb cellular signaling and functions. For example, enteroviruses rapidly inhibit cap-dependent host mRNA translation 4 , 5 . Traditionally, this activity was considered to be important for redirecting resources for optimal translation of viral RNA (vRNA), but this activity may also be important to suppress de novo synthesis of proteins with antiviral function 4 , 6 . Moreover, enteroviruses disturb the integrity of the nuclear pore complex (NPC), thereby triggering a so-called nucleocytoplasmic trafficking disorder (NCTD). This activity results in the translocation of cellular RNA binding proteins to the cytosol, such as La-autoantigen and Pyrimidine-tract binding protein (PTB) 7 – 9 , which are thought to enhance key viral processes such as vRNA translation and genome replication. Moreover, it results in the nuclear retention of host mRNA, proposedly to suppress cellular antiviral signaling 6 , 10 . Furthermore, enteroviruses suppress the formation of stress granules (SGs), which are considered to serve as a platform for the assembly and integration of several antiviral signaling pathways, including the integrated stress response (ISR) and type I interferon response (IFN-α/β) pathways. Apart from this, enterovirus infection results in the cleavage of dsRNA sensors and downstream signaling molecules involved in the induction of IFN-α/β production. Both 2A pro and 3C pro have been attributed central roles in the viral strategies to optimize vRNA translation and replication, and antagonize the host antiviral responses described above. However, as of yet, we know little about the relative importance and individual contribution of each protease to these strategies. Most studies were conducted using either in vitro experiments, ectopic overexpression, or surrogate infection models, such as encephalomyocarditis virus (EMCV) carrying inactivating mutations in the Leader (L) protein (EMCV-L(Zn)) expressing 2A pro as a heterologous protein. Unfortunately, the ectopic expression of the protease isolated from all other viral proteins does not accurately reflect the context and spatio-temporal dynamics of infection. Although the surrogate system does entail an infection scenario, it is of another virus and leaves open the question of whether the observed effects occur in EV-infected cells and if they are relevant. Thus, the study of viral proteins in isolation or out of the infection setting of their parent virus can give erroneous or difficult-to-interpret insights into their true function. This is exemplified by studies towards the function of the L protein of Crimean Congo Hemorrhagic Fever virus (CCHFV), whose predicted deubiquitinase (DUB) domain was shown to inhibit IFN signaling when expressed in isolation, but upon inactivation in the virus was identified to be involved in cleaving off ubiquitin in cis from L to regulate its function, rather than to suppress IFN signaling 11 . The critical role that both proteases play in polyprotein processing has largely precluded the possibility of catalytically inactivating them, and thereby to study their individual contribution to the virus-host interaction in the relevant infection context. Previously, mutants of PV and EV-A71 were reported where 2A pro had been either removed entirely or catalytically inactivated, enabled in both cases by the introduction of a second IRES in between P1-P2 12 , 13 . This resulted in severe attenuation, but other than assessing the role of 2A pro for SG formation 12 , these viruses were not characterized in great detail. Here, we report the construction of viable CVB3, in which we replaced the 2A pro cleavage site at the VP1/2A junction with either an additional IRES, a T2A self-cleaving peptide, or a 3CD cleavage site (3CD cs ), enabling mutational inactivation of 2A pro . CVB3 containing a catalytically-inactive 2A pro (CVB3-2Amut) exhibits only a relatively minor reduction in fitness in some cell types, but is nearly completely restricted in others. Furthermore, CVB3-2Amut is severely attenuated in vivo in a CVB3 pancreatitis mouse model, illustrating the important role that 2A pro plays during infection. Detailed characterization of the consequences of 2A pro inactivation in CVB3 infected cells reveals that 2A pro , but not 3C pro , plays key roles in suppressing host mRNA translation, NCTD induction, suppressing SGs, and inhibiting IFN-α/β responses. Employing VIRIM (Virus Infection Real-time Imaging), an advanced single-molecule live cell imaging assay that we recently developed 14 , we show that 2A pro inactivation affects the dynamics of the early phases of the enterovirus life cycle and interferes with the ability to progress through an early critical phase, namely replication of the incoming viral genome. In conclusion, we identify the distinctive role of 2A pro in promoting viral translation, replication, modification of host cell functions, and suppression of antiviral responses in enterovirus-infected cells. Results Design, generation, and growth kinetics of recombinant CVB3 with inactive 2A pro To engineer recombinant CVB3 viruses with catalytically inactive 2A pro , we replaced the 2A pro cleavage site at the P1-P2 junction with either (i) the IRES sequence of Theiler’s encephalomyelitis virus (TMEV), (ii) a self-cleaving peptide from Thosea asigna virus (T2A), or (iii) a sequence corresponding to the 3C/3D cleavage site recognized by 3C pro (3CD cs ) ( Fig. 1a ). All these elements effectively mediated separation of P1 and P2, alleviating the critical role of 2A pro in polyprotein processing and allowing for its catalytic inactivation. Specifically, mutations were introduced in the catalytic triad (H21A, D39A, and C110A) to abolish 2A pro activity (these will further be referred to as 2Amut). Recombinant CVB3 viruses were recovered upon transfection of human embryonic kidney 293-T (HEK293-T) cells with in vitro RNA transcripts obtained from cDNA clones. Download figure Open in new tab Fig. 1: Design and characterization of Coxsackievirus B3 with catalytically-inactive 2A protease (2A pro ). ( a ) Schematic representation of the wild-type (wt) Coxsackievirus B3 (CVB3) genome and its engineered derivative viruses with inactivated 2A pro (2Amut). The 2A pro cleavage site was replaced by a Theiler’s murine encephalomyelitis virus (TMEV)-derived IRES sequence, a T2A peptide, or a sequence corresponding to the cleavage site of 3C/3CD protease (3C pro ) (3CD cs ). Specific cleavage sites of the viral proteases are indicated with blue (2A pro ) and green (3C pro ) triangles. ( b ) Growth kinetics of recombinant 2A pro wt viruses (3CDcs-2Awt, T2A-2Awt and IRES-2Awt) in HeLa-R19 cells infected at a multiplicity of infection (MOI) of 5, as measured by TCID 50 assay (upper panel) or qPCR (lower panel). ( c ) Growth kinetics of the different 2Awt and 2Amut viruses in HeLa-R19 cells. Experiment was performed as in panel (b). ( d ) HeLa-R19 or HEK293-T cells were transfected with equal amounts of in vitro transcribed RNA of Renilla-luciferase (RLuc)-3CDcs-2Awt or -2Amut, and luciferase levels were determined at the indicated times post infection. ( e ) Growth curves of 2Awt and 2Amut viruses obtained from infected HeLa-R19, BGM, Vero E6 and A549 cells. Data represent the mean ± SEM of one representative experiment (b, c, d) or three independent experiments (e). To assess the growth kinetics of the CVB3 with inactive 2A pro , single-cycle growth curves were performed on HeLa cells, measuring both infectious particles (TCID 50 /ml) and vRNA levels. All three 2A-wild type (wt) viruses replicated well, albeit with a slight reduction in resulting infectious virus titers compared to wt-CVB3, especially for CVB3-T2A-2Awt. This suggests that the introduction of alternative elements at the P1-P2 junction generally effects separation, yet with somewhat differing efficiency ( Fig. 1b ). Notably, all CVB3-2Amut viruses showed a delay in vRNA replication and a reduction in virus production compared to 2Awt viruses ( Fig. 1c ). To investigate in more detail the impact of 2A pro inactivation on vRNA translation and replication, we transfected HeLa and HEK293-T cells with equivalent amounts of in vitro transcribed RNAs from CVB3-3CD cs -2Awt and 2Amut cDNAs containing the Renilla luciferase (RLuc) upstream of the capsid coding region. The increase in luciferase signal that can be observed after 5 hours (hrs), which reflects translation of newly synthesized vRNAs, was severely reduced by 2A pro inactivation ( Fig. 1d ). In contrast, no large differences were observed in the luciferase levels produced in the first hrs after transfection, before substantial replication has taken place, implying that 2A pro has little effect on the efficiency of translation on the incoming vRNA. Together, these data highlight the essential role of the proteolytic activity of 2A pro for efficient vRNA replication and subsequent virus production. The somewhat modest reduction in viral growth observed in HeLa cells prompted us to explore whether this might be more pronounced in different cell lines. To this end, we first titrated our CVB3-3CD cs and T2A virus stocks on various cell lines and assayed the development of cell death. Comparison of CVB3-3CD cs -2Awt and -2Amut revealed that, whereas 2A pro -inactivated virus replicated relatively well in HeLa cells, replication in Buffalo green monkey (BGM) kidney cells, African green monkey kidney Vero-E6 cells, and human lung carcinoma A549 cells was severely impaired ( Supplementary Fig. 1a ). Consistently, infectious virus production was shown to be severely restricted on BGM, Vero, and A549 when assessed by titration on susceptible HeLa cells ( Supplementary Fig. 1b ). As we observed only minor differences between the CVB3-IRES, - 3CD cs , and -T2A viruses, we used the CVB3-3CD cs- 2Awt and corresponding 2Amut viruses in further experiments. These viruses will further be referred to in the text as CVB3-2Awt and CVB3-2Amut, respectively. 2Apro activity is indispensable for CVB3 virulence and replication in a murine model The more severe attenuation we observed in certain cell lines with CVB3-2Amut prompted us to investigate the virulence of this mutant virus in an in vivo setting. To this end, we employed a well-established CVB-induced pancreatitis mouse model 15 , 16 . SJL mice ( n = 4 per group) were infected intraperitoneally with equivalent amounts (either 1.0*10 7 TCID 50 /ml or 5.0*10 5 TCID 50 /ml) of CVB3, CVB3-2Awt, or CVB3-2Amut. At 3 days postinfection - i.e., the time point at which a peak of pancreatic lesions can be observed - the mice were sacrificed and the pancreas was removed for analysis ( Fig. 2a ). Infectious virus titers were determined by endpoint titration and vRNA levels quantified using RT-qPCR. Furthermore, using a standardized scoring system, we assessed histopathological damage (i.e., necrosis, inflammation, and edema). Virus titers and vRNA levels in the pancreas of mice infected with CVB3-2Awt were lower (10- or 100-fold, respectively) and histopathological damage was less severe than in mice infected with CVB3 ( Fig. 2b-c ). This may be explained by a delay in replication of CVB3-2Awt in some cell types, as observed in A549, BGM, and Vero cells ( Fig. 1b ). CVB3-2Amut, however, was severely impaired in replication. Infection with this virus resulted in 1000-fold less vRNA in the pancreas compared to CVB3-2Awt, no infectious virus could be isolated from the pancreas, and no histopathological damage was observed. Thus, CVB3-2Amut is severely attenuated in vivo , emblematic of the pivotal role which 2A pro plays in many aspects of the viral life cycle. To unravel this further, we resorted to studying CVB3-2Amut in HeLa cells, as this cell line supported replication relatively well. Download figure Open in new tab Fig. 2: 2A pro activity is essential for CVB3 replication and pathogenesis in vivo. ( a ) Schematic representation of the experimental procedure. SJL mice ( n =4 per group) were infected intraperitoneally with equivalent viral doses (either 1.0*10 7 TCID 50 /ml or 5.0*10 5 TCID 50 /ml) of CVB3, CVB3-2Awt or CVB3-2Amut. Mice were sacrified three days post-infection for pancreas isolation and processing. ( b ) Viral RNA levels and infectious viral titers in the pancreas of infected mice were determined by RT-qPCR and end-point titration, respectively. ( c ) Pancreatic histopatology was assessed using a standardized scoring system ranging from 0 to 4 for necrosis (0 = not affected, 1 = <10% of tissue affected, 2 = 11-25% of tissue affected, 3 = 26-50% of tissue affected, 4 = more than 50% of tissue affected), and from 0 to 3 for inflammation and edema (0 = none, 1 = mild, 2 = moderate, 3 = severe). Analyses were conducted without knowledge of the experimental groups. Statistical signifance was determined by Mann-Whitney U test. Differences were considered significant if p<0.05. 2Apro is essential for disrupting nucleocytoplasmic trafficking in CVB3-infected cells Enteroviruses profoundly disrupt active nucleocytoplasmic transport, which is regulated by the NPC, resulting in a NCTD. Both the classical NLS, M9-NLS, RS-NLS, and classical NES pathways are affected 6 , leading to the cytosolic relocalization of several nuclear-resident host factors, e.g., RNA binding proteins, to enhance viral RNA translation and/or replication 6 . Furthermore, this results in the defective transport of (signaling) proteins into the nucleus and a block in mRNA export, thereby impairing antiviral response activation 10 , 17 . Both 2A pro and 3C pro have been implicated in triggering the NCTD by cleaving several phenylalanine-glycine (FG)-repeat-nucleoporins, which interact with each other to maintain the permeability barrier of the NPC. The initial induction of the NCTD correlates temporally with the cleavage of Nup98, which is mediated exclusively by 2A pro 17 – 21 . Later during infection, other Nups have been reported to be targeted as well by both 2A pro (Nup62, Nup153, and Nup358) 10 , 17 , 19 , 21 – 25 , and by 3C pro (Nup153, Nup214, and Nup358) 20 , 26 , but the extent to which these contribute to the NCTD is unknown. To assess the relative importance of 2A pro for causing the NCTD, we performed Western blot (WB) analysis ( Fig. 3a ). Cleavage products of Nup98 were detected in CBV3 and CBV3-2Awt infected cells from 2 hours post infection (hpi) onwards, with complete cleavage at 6 hpi. CVB3-2Amut was unable to cleave Nup98, even at later time points ( Fig 3a ), in agreement with earlier observations that 3C pro does not cleave this Nup 20 . Similarly, using the general anti-FG-nup mAb-414 we demonstrate that 2A pro cleaves a host of other FG-Nups, including Nup62, Nup153, and Nup214, but this occurs only at later time points 10 , 22 , 23 . Moreover, we failed to observe any cleavage of Nups at the hands of 3C pro during CVB3-2Amut infection. Download figure Open in new tab Fig. 3: 2A pro plays a critical role in distrupting nucleocytoplasmic trafficking in CVB3 infected cells. ( a ) Western blot analysis for Nup98, FG-repeat-containing nucleoporins, CVB3 2C protein and β-actin. HeLa-R19 cells, (mock-)infected with CVB3, 3CD cs -2Awt, or 3CDc cs -2Amut at an MOI of 10, were lysed at 2, 4, 6, 8 and 10 hpi. Black triangles denote intact proteins, white triangles indicate cleavage products, asterisk refers to a non-specific band which has been observed before with this antibody 18 . ( b ) Nucleocytoplasmic trafficking disorder (NCTD) is abolished in 3CD cs -2Amut infected cells. HeLa-3xNLS-GFP were infected at MOI of 10 with CVB3, 3CD cs -2Awt, and 3CD cs -2Amut. Cells were fixed with 4% paraformaldehyde at 2, 4, 6, 8, 10 hpi and analyzed by IF analysis for dsRNA as a marker of infection, for Nup98 as marker of the NPC, for hnRNPK and 3xNLS-GFP localization as indicators of NCTD. Scale bar: 25 μm. To corroborate the correlation between 2A pro -mediated Nup cleavage and the induction of NCTD, we performed immunofluorescence (IF) analysis on HeLa cells stably expressing green fluorescent protein (GFP) fused to three tandem nucleus localization signals (HeLa-3xNLS-GFP) ( Fig. 3b ). Cells infected with CVB3-2Awt showed rapid leakage of 3xNLS-GFP and heterogeneous nuclear ribonucleoprotein K (hnRNPK), a nuclear resident protein, from the nucleus to the cytosol. Moreover, we observed an altered nuclear morphology and less pronounced Nup98 signal in the nuclear rim over time, which was not observed in cells infected with CVB3-2Amut, emphasizing the essential role of 2A pro in inducing NCTD and the dispensable contribution of 3C pro . Of note, the failure to trigger NCTD in the CVB3-2Amut-infected cells is not due to a lack of 3C pro activity, as evidenced by the observation of viral protein expression in CVB3-2Amut-infected cells from 4 hpi, albeit it at a lower level than in CVB3-2Awt-infected cells ( Fig. 3a ), and from the observation of other 3C pro -mediated cleavage events, such as that of Ras GTPase-activating protein-binding protein 1 (G3BP1) and RIG-I, in the same experimental lysates (see Fig. 4d and Fig. 6a ). Together, our data present evidence that 2A pro plays a critical role in triggering the NCTD in infected cells and argue that the 3C pro -mediated cleavage activities have little if any effect on nucleocytoplasmic transport. Download figure Open in new tab Fig. 4: 2A pro , but not 3C pro , selectively inhibits host translation and suppresses stress granule formation. ( a ) Western-blot analysis of eIF4GI, DAP5, PABP, PKR, p-PKR and p-eIF2α. Cell lysates correspond to the same experiment shown in Fig. 3a . Black triangles denote intact proteins, white triangles indicate cleavage products. ( b ) THRONCAT in vivo metabolic labeling of HeLa-R19 cells infected with 3CD cs -2Awt and 3CD cs -2Amut. Cells treated with sodium arsenite served as a positive control for translation arrest. Lysates were run on SDS-PAGE gels and subsequently transfered to nitrocellulose membranes for analysis. ( c ) Representative images (left) and quantification (right) of SG formation in HeLa-R19 cells infected at an MOI of 10 with CVB3, 3CD cs -2Awt or 3CD cs -2Amut. Cells were fixed at 4, 6, 8, 10 hpi and analyzed by IF analysis for dsRNA as a marker of infection and eIF3 and G3BP2 as SG markers. Magnified regions are shown in white boxes. Scale bar: 25 μm. Quantification of small blue and large purple SG formation is shown on the right. At least 1000 cells were analyzed for each virus and time point. Small SG positivity is given as a percentage of total cells, since most cells are not yet positive for dsRNA at early timepoints. ( d ) Western blot analysis of G3BP1, CVB3 2C protein, and β-actin. HeLa-R19 cells, either mock-infected or infected with recombinant CVB3s at an MOI of 10, were lysed at 6, 8, 10, 12 and 16 hpi. Black triangles denote intact proteins, empty triangles indicate cleavage products. 2A pro , but not 3C pro , mediates the selective shutdown of host translation A hallmark of enterovirus infection is the selective shutdown of host cap-dependent translation initiation. 2A pro plays a major role in this shutdown by cleaving eIF4G, which is essential for 5’-cap dependent translation initiation but not for viral IRES-dependent translation initiation 27 – 31 . In fact, the C-terminal cleavage fragment of eIF4G has been implicated in enhancing vRNA translation 32 – 34 . Another cellular protein, death-associated protein 5 (DAP5), which is structurally homologous to the eIF4G C-terminal cleavage fragment, is suggested to drive viral translation initiation during the first round of translation before substantial eIF4G cleavage 34 . As infection progresses, 2A pro cleaves DAP5, resulting in a DAP5-N fragment that can still support IRES-mediated vRNA translation and a DAP5-C fragment that negatively affects cap-dependent translation 35 . Additionally, both 2A pro and 3C pro have been shown to cleave poly-(A)-binding protein (PABP), which is crucial for mRNA stability and translation initiation. However, the degree to which this contributes to the host shut-off is unclear. To examine the importance of 2A pro for the virus-induced host shut-off, we assessed eIF4G, DAP5, and PABP cleavage by WB at different time points post-infection. As expected, eIF4G cleavage products became apparent as early as 2 hpi in cells infected with wt 2A pro viruses. In contrast, the cellular eIF4G pool remained fully intact during 2Amut infection, even at later time points ( Fig. 4a ). Similarly, DAP5 cleavage – detected from 6 hpi onward in CVB3-2Awt-infected cells – was absent in CVB3-2Amut-infected cells, even at later time points ( Fig. 4a ). Cleavage of PABP, which we assessed using the monoclonal antibody 10E10 that only detects full-length PABP but not cleavage products, was observed in cells infected wt 2A pro viruses, but not in cells infected with CVB3-2Amut ( Fig. 4a ), not even at 16 hpi ( Supplementary Fig. 2a ). To evaluate the ability of CVB3 lacking 2A pro activity to induce host translation shut-off, we performed in vivo metabolic labeling using threonine-derived non-canonical amino acid tagging (THRONCAT) 36 , a novel method that uses ethynylserine as a biorthogonal threonine analogue to mark newly synthesized proteins ( Fig. 4b ) 36 . Infection of HeLa cells with CVB3-2Awt resulted in a progressive decrease in general protein synthesis starting at 3 hpi, with selective viral translation indicated by the appearance of specific bands representing viral proteins. Conversely, no such early selective translation shut-off was observed for CVB3-2Amut, but rather from 6 hpi a seemingly complete shut-off of all translation was observed, reminiscent of activation of the ISR (as exemplified by Arsenite treatment of cells, see lane 2). Notably, viral IRES-dependent translation has been suggested to be rendered independent of eIF2α (and thereby eIF2α phosphorylation) by 2A pro , via a still unknown mechanism 37 , 38 . Due to the reduced replication efficiency of CVB3-2Amut and likely the inability to counter the ISR, viral proteins are poorly detectable in the background of cellular proteins. Activation of the ISR in enterovirus-infected cells is indicated by the phosphorylation of one of the ISR kinases, dsRNA-activated protein kinase (PKR), and the ensuing phosphorylation of alpha subunit of translation initiation factor eIF2 (eIF2α) 37 , 12 , 39 – 45 . Interestingly, we observed earlier and more pronounced PKR and eIF2α phosphorylation in CVB3-2Amut-than in CVB3-2Awt-infected cells ( Fig. 4a ). This observation is remarkable as, due to the delayed replication, there is less dsRNA formed in CVB3-2Amut-infected cells. Cleavage of PKR by 3C pro , a well-known phenomenon observed in enterovirus-infected cells 43 , was observed upon both CVB3-2A-wt and CVB3-2Amut infection ( Fig. 4a ). However, this cleavage occurred only late in infection, after PKR activation and eIF2α phosphorylation, and thereby likely has little, if any, effect on ISR activation. Instead, our data suggest that 2A pro may delay PKR phosphorylation and ISR activation. Critical role of 2A pro in counteracting stress granule formation during enterovirus infection Despite ISR activation, enteroviruses effectively suppress downstream canonical SG formation 12 , 46 , 47 . Initially, it was implied that 3C pro of both PV and EMCV, by cleaving G3BP1, a key SG nucleation factor, played a central role in this 48 , 49 . However, this has been challenged by the observation that 3C pro , when expressed as a heterologous protein in the EMCV-Zn surrogate infection model, is unable to suppress SG formation, despite substantial G3BP1 cleavage. In contrast, 2A pro potently suppressed SG formation in the same system 47 . Yang and colleagues showed during EV-A71 infection that it is indeed 2A pro which renders the infected cells progressively more resistant to SG formation, and that this is conserved among different enterovirus species 12 . Of note, this was shown to rely on the catalytic activity of 2A pro , rather than eIF4G cleavage specifically 50 . We performed IF analysis to assess SG formation during CVB3-2Amut infection, staining for the conventional stress granule markers G3BP2 and eIF3 ( Fig. 4c ), as well as TIA-1 and Sam68 ( Supplementary Fig. 2b ). In line with earlier findings 12 , 46 , CVB3-2Awt induces small SGs in the early phase of infection (4 hpi), which disappear at later time points 12 , 47 . These are thought to be the consequence of the 2A pro -mediated cleavage of eIF4G and the ensuing inhibition of cap-dependent host mRNA translation. We find these early SGs to be positive for G3BP1 during CVB3 infection, whereas reports from other groups have observed them to be both positive in the case of CVB3 46 and negative in the case of EV-A71 12 for G3BP1. In line with the putative role of eIF4G cleavage, CVB3-2Amut did not induce the early appearance of small SGs. From 6 hpi onwards, however, CVB3-2Amut triggered the formation of large SGs containing eIF3, G3BP2 and TIA-1, but not Sam68. Previously, both we and others have shown that these SGs are dependent on the PKR-mediated phosphorylation of eIF2α 12 , 47 . Importantly, we still observe substantial cleavage of G3BP1 during CVB3-2Amut infection, consistent with continued 3C pro activity ( Fig. 4d ). In conclusion, despite the cleavage of PKR and G3BP1 in CVB3-3CD cs -2Amut virus infected cells, SGs were not suppressed. Thus we confirm that CBV3 2A pro , rather than 3C pro , is critical for SG suppression during infection. Differences in cell morphology and death between CVB3-2Awt and -2Amut infection 2A pro has been reported to cleave cytoskeletal components, such as cytokeratin 8 and dystrophin 51 , 52 , and was shown to trigger apoptosis when individually expressed 53 . Therefore, we examined differences in cell morphology and cell death between cells infected with CVB3-2Awt and -2Amut using fluorescence microscopy ( Fig. 5a ). Cells infected with CVB3-2Awt exhibited profound morphological changes, including cytoplasmic shrinkage, cytoskeleton rearrangement, indicated by β-tubulin staining, and altered nuclear shape. In contrast, cells infected with 2Amut virus showed morphological changes only at much later stages of infection, with apoptotic-like bodies and nuclear fragmentation appearing from 10 hpi onwards ( Fig. 5a-b ). Importantly, neither of these was clearly observed during CVB3-wt infection. Furthermore, cells infected with CVB3-2Amut generally remained more attached and retained their stretched morphology throughout infection, with cell shrinkage and rounding occurring only much later compared to cells infected with CVB3-2Awt, despite the presence of large amounts of viral proteins and RNA. This is in line with the marked absence of clear CPE observed in cells infected with CVB3-2Amut until very late in infection. Download figure Open in new tab Fig. 5: Distinct effects of CVB3-2Awt and CVB3-2Amut on cell morphology and death. ( a ) HeLa-R19 cells were infected with 3CD cs -2Awt and 3CD cs -2Amut at an MOI of 50, fixed at 6, 8, 10, 12 and 16 hpi and, and analyzed by IF analysis. Nuclei were stained with DAPI, dsRNA was detected as marker for infection, and β-tubulin was used as a cytoskeleton marker. Fragmented nuclei are indictaed by white triangles. Scale bar: 20 μm. ( b ) Quantification of infected cells displaying nuclear fragmentation in panel a. ( c ) Western blot analysis of caspase 7 (Casp7) and PARP using cell lysates corresponsing to the same experiment shown in Fig. 4d . Black triangles mark intact proteins, white triangles the corresponding cleavage products. Download figure Open in new tab Fig. 6: 2A pro is the key viral protease responsible for the suppression of type I interferon signaling. ( a ) HeLa-R19 cells were infected at an MOI of 10 and lysed at 2, 4, 6, 8, 10 and 16 hpi. Total RNA was analyzed by RT-qPCR for IFN-β, IFIT1, viral RNA (vRNA) and actin. IFN-β, IFIT1 and vRNA levels were calculated as fold induction compared to levels in mock-infected cells, after correction for actin mRNA levels, and normalized to the expression levels at 2 hpi. Data represent mean ± SEM of three technical replicates. Statistical significance was calculated by multiple two-tailed unpaired t-tests; * p<0.5 ; ** p<0.01 ; *** p<0.001. ( b ) Western blot analysis for MDA5, RIG-I, MAVS, IF3 and p-IRF3. Cell lysates belong to the same experiment shown in Fig. 3a . Black triangles point out intact proteins, white triangles indicate the cleavage products. ( c ) Hela-R19 cells were pretreated for 16 hrs with IFN-α and subsequently infected with RLuc-3CD cs -2Awt and -2Amut viruses. Cells were lysed at 6 hpi and luciferase levels were assessed. Data represent the mean ± SEM of three independent experiments. WB analysis revealed markers of apoptosis induction, namely caspase-7 (Casp7) and poly ADP-ribose polymerase (PARP) cleavage, in cells infected with CVB3, irrespective of 2A pro catalytic status ( Fig. 5c ). Notably, we detected cleavage of Casp7 and PARP in CVB3-2Amut infected cells again only at much later time points. In conclusion, CVB3 infection seems to trigger apoptotic signaling, as shown by Casp7 and PARP cleavage. However, 2A pro catalytic activity appears essential to prevent the cellular morphological changes and nuclear fragmentation, normally associated with canonical apoptosis. 2A pro , but not 3C pro , is essential to suppress type I IFN signaling Enterovirus genome replication inevitably results in the formation of long dsRNA replication intermediates that are primarily recognized by the cytosolic RLR melanoma differentiation-associated protein 5 (MDA5) 54 . RIG-I, which recognizes dsRNA with 5’-triphosphate groups, which are absent on picornavirus genomic RNA, was found to be dispensable for sensing picornavirus infection in several studies 54 – 56 , although a role of RIG-I in sensing EMCV and CVB3 infection in mouse macrophages has been suggested 57 . Extensive work has shown that both 2A pro and 3C pro cleave key components of the RLR pathway. 2A pro cleaves MDA5 and MAVS, whereas 3C pro targets RIG-I and MAVS 58 – 61 , 61 – 63 . However, in an EMCV-L(Zn) surrogate infection model, 2A pro , rather than 3C pro , mediated effective suppression of IFN-β responses 47 . To evaluate the respective roles of 2A pro and 3C pro in suppressing the RLR signaling pathway and IFN-α/β interferon production during CVB3 infection, we conducted qPCR experiments to measure mRNA levels of IFN-β and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), an interferon-stimulated gene (ISG) product that can also be induced directly by phosphorylated IRF3. In line with the results shown in ( Fig. 1 ), we observed a decrease in viral RNA copies during infection with CVB3-2Amut ( Fig. 6a ). Despite this slower replication, and in contrast to CVB3-2Awt, infection with CVB3-2Amut resulted in significant induction of both IFN-β and IFIT1 gene transcription, from 6 hpi onwards ( Fig. 6a ). To gain more insight into the underlying mechanism, we performed WB analysis to examine the cleavage of MDA5, RIG-I and MAVS, alongside phosphorylation of the downstream transcription factor IRF3. We observed cleavage of both MDA5, MAVS, and RIG-I from 6 hpi onwards during CVB3-2Awt infection ( Fig. 6b ). Importantly, we detected no IRF3 phosphorylation, even at later time points. In contrast, no cleavage of MDA5 and MAVS was observed in cells infected with CVB3-2Amut up to 10 hpi. Only at later time points, a MAVS cleavage fragment was observed of similar size as previously reported upon cleavage by 3C pro ( Supplementary Fig. 3a ). We still detected cleavage of RIG-I during CVB3-2Amut infection, albeit at later time points (i.e., from 8 hpi onwards). Despite this, IRF3 phosphorylation could be detected from 8 hpi onwards, consistent with transcriptional activation of the IFN response observed earlier. These data provide evidence that 2A pro , and not 3C pro , is the main antagonist of the IFN-α/β response during CVB3 infection. Aside from its central role in suppressing RLR signaling and subsequent IFN production, 2A pro may also be important for enteroviruses to overcome the antiviral state established in cells via paracrine IFN-α/β signaling 64 . Previously, it was shown that the addition of the poliovirus 2A pro gene to the EMCV genome allowed EMCV to replicate in IFN-alpha-pretreated cells 64 . To assess if CVB3, in the absence of 2A pro activity, is unable to overcome IFN pretreatment, we performed luciferase assays on cells pretreated with IFN-α2, infected with renilla-luciferase (RLuc) variants of CVB3-2Awt and -2Amut. We observed that whereas CVB3-2Awt replication remained largely unaffected, CVB3-2Amut was highly sensitive to pretreatment with IFN-α2, with progressively more inhibition at higher concentrations ( Fig. 6C ). This confirms that 2A pro is critical to overcome the antiviral state induced by IFN signaling. Single-molecule imaging to elucidate the dynamics of CVB3-2Amut translation and replication To gain more insight into the delay in replication that we observed for CVB3-2Amut, we turned to a single cell imaging assay recently developed in our lab termed VI rus Infection R eal-time IM aging (VIRIM) 14 . Briefly, VIRIM is a two-component system consisting of cells expressing a single-chain variable antibody fragment fused to a fluorescent protein (SunTag-antibody, STAb-FP) and viruses that have been genetically engineered to express a short suntag peptide array at the N-terminus of the viral polyprotein followed by a 3C pro cleavage site ( Fig. 7a ). Upon translation of the viral genome in STAb-FP expressing cells infected with Suntag-CVB3 (ST-CVB3), STAb-FP will co-translationally bind to the suntag peptides emerging from the ribosome ( Fig. 7b ). Since multiple ribosomes simultaneously translate a single vRNA, many STAb-FPs will be recruited to vRNAs undergoing translation, and these translating vRNAs can be observed as bright fluorescent foci using spinning-disk confocal microscopy. Importantly, cessation of translation results in the release of individual ‘mature’, STAb-FP-bound SunTag peptides with their associated fluorescence from the vRNA. Such mature SunTag peptides are subsequently dispersed throughout the cytoplasm, where they are too dim to be observed as fluorescent foci. Therefore, fluorescent foci specifically mark sites of active translation. As vRNA translation and replication occur in opposite directions on the same vRNA strand, they are temporally exclusive. Consequently, translation necessarily ceases upon the start of replication, leading to a disappearance of VIRIM signal ( Fig. 7b ). Thus, by analyzing the number of VIRIM foci over time in a single infected cell, we can identify distinct phases of translation and replication of CVB3 during early infection ( Fig. 7c,d ) 14 . Download figure Open in new tab Fig. 7: Single-molecule imaging to dissect the role of 2A pro during the earliest phases of CVB3 infection. (a) Schematic representation of the SunTag-CVB3 viruses. 5xSunTag cassette flanked at its 3’-side by a 3CD cs was inserted before the P1 region of CVB3, 3CD cs -2Awt, or 3CD cs -2Amut. ( b ) Schematic representation of the VIRIM assay. Left panel shows active translation. Right panel denotes situation during viral replication. The blue region of the viral genome indicates the SunTag peptide sequences. Note the opposing directions of both processes, and their consequent temporal exclusiveness. ( c ) Schematic representation of the different phases of early enterovirus infection as defined by Boersma et al. 2020. ( d ) Pictures of the different phases of viral infection, as observed for representative infections with 3CD cs -2Awt, and 3CD cs -2Amut, respectively. The time since start phase 1 is stated in grey. The numer of translating vRNAs in the infected cell is stated in red. Schematic illustration of VIRIM spot number is shown in the top panel. The far right graphs show VIRIM spot count for the infections shown in the representative images on the left. ( e ) Kaplan-Meier curves depicting the duration of the different phases of infection for CVB3, 3CD cs -2Awt, and 3CD cs -2Amut. ( f ) The average number of spots observed over time during CVB3, 3CD cs -2Awt, and 3CD cs -2Amut infection. Shaded areas represent SEM. ( g ) Violin plots depicting the average rate of increase in the number of translating vRNAs during phase 3 (phase 3 slope) (left), and the average number of translating vRNAs in phase 4 (phase 4 plateau, see also ( c )) (right). Scale bar 10 μm. We engineered CVB3-2Awt and -2Amut to encode a 5x-SunTag (ST) array before VP4. Recovered viruses stably maintained the insert over passage. During time-lapse imaging of infected SunTag cells additionally expressing a blue fluorescent protein-(BFP-)NLS NCTD reporter construct (as described previously 14 ), we observed leakage of the reporter construct from the nucleus for ST-CVB3-2Awt, starting within minutes and being complete within the first 2h after initial translation was detected ( Supplementary Fig. 4 ). Consistent with our earlier data, BFP-NLS remained confined to the nucleus in cells infected with ST-CVB3-2Amut, even at late times post infection ( Supplementary Fig. 4 ). We could discern the same general phases for both ST-CVB3-2Awt and -2Amut as we previously observed for ST-CVB3-wt infections ( Fig. 7e ) 14 . We observed the appearance of a single spot upon translation of the incoming vRNA (phase 1), which disappeared as result of cessation of translation and the start of replication (phase 2), after which new foci appeared due to translation of newly synthesized vRNAs (phase 3), followed by translation of newly-synthesized vRNAs (phase 4) and finally and second wave of replication and translation that results in a further increase in VIRIM foci (phase 5) ( Fig. 7e ) . The duration of phases was similar for ST-CVB3-2Awt and -2Amut infections, with no apparent differences in timing of either translation or replication ( Fig. 7e ). However, we observed significantly less VIRIM foci reappearing in phase 3 and beyond during ST-CVB3-2Amut infection ( Fig. 7f,g ), implying replication was less efficient. Moreover, a substantially higher fraction of infections were arrested during initial replication of ST-CVB3-2Amut ( Fig. 7e ). This supports our earlier observations that initial replication constitutes the most vulnerable phase of infection 14 . Moreover, it illustrates the potency and importance of 2A pro catalytic activity to facilitate the very first round of replication. Of note, we observed a somewhat higher phase 2 arrest rate for ST-CVB3-wt compared to what we observed previously, i.e., ∼20-25% 14 , which is likely due to the use of another monoclonal cell line in the current study. In accordance with previous results, we observed that the duration of phase 1 (initial translation), was somewhat extended in cells with unsuccessful replication. Importantly, this held true regardless of 2A pro catalytic activity ( Supplementary Fig. 4C ). We previously observed that for a fraction of infections during which the first round of replication is not successful, viral translation reinitiates on the incoming vRNA, after which there is another attempt at replication. These translation pulses and replication breaks can occur multiple times, and can either culminate in replication success or arrest 14 . Whereas we previously observed that the number of pulses and breaks increases starkly upon disruption of viral replication (with a 3D pol inhibitor), no such increase was observed for ST-CVB3-2Amut ( Supplementary Fig. 4D ). 2Apro progressively stimulates viral translation efficiency during the course of infection VIRIM can also be used to assess viral translation efficiencies. The C-terminal cleavage product of eIF4G stimulates enteroviral IRES-mediated translation 32 – 34 . Additionally, nuclear-resident RNA-binding proteins, which are relocalized to the cytosol due to the NCTD, have also been proposed to stimulate enteroviral translation 6 . Therefore, we set out to investigate whether, because of the absence of 2A pro -mediated eIF4G cleavage and NCTD, viral translation would be progressively less efficient over the course of CVB3-2Amut infection. VIRIM translation spot intensity is dictated by the number of SunTag-containing nascent chains associated to ribosomes that are translating a vRNA, and hence by the efficiency of viral translation initiation ( Supplementary Fig. 5a ). Thus, to compare translation efficiency over time, we measured the intensity of VIRIM translation foci within single cells infected with either ST-CVB3-2Awt or -2Amut. During ST-CVB3-2Awt infection, we observed that spot intensity on average increased from phase 1 to phase 4. However, no such significant increase could be observed for 2Amut infections ( Fig. 8a ). These results are in line with a progressive increase in translation efficiency over the course of infection with CVB3 carrying a proteolytically active 2A pro , brought about by the diverse and critical actions of 2A pro , and might (partially) underly the decrease in the efficacy of replication in ST-CVB3-2Amut infected cells. Importantly, during phase 1, when no significant cleavage of eIF4G has yet occurred, there were no significant differences in translation efficiency between ST-CVB3-2Awt and ST-CVB3-2Amut ( Fig. 8b ). The higher rate of phase 2 arrest is therefore unlikely the consequence of a lower translation efficiency during phase 1, but rather suggests a direct role of 2A pro in the first round of viral replication. Download figure Open in new tab Fig. 8: The increase in viral translation efficiency during infection depends on the action of 2A pro . ( a ) VIRIM spot intensity was measured during phase 1 and phase 4 in the same infected cells. Spot intensities are plotted relative to phase 1 spot intensity. Each line and red dot represents an individual infected cell. Median values are indicated above the graphs. ( b ) Normalized intensity (AU) of phase 1 translation spots during infection with CVB3, 3CD cs -2Awt, and 3CD cs -2Amut. The higher intensity of the 3CD cs viruses can be explained by the comparatively longer effective transcript length (i.e., the length of vRNA that is decoded by the ribosome before the SunTag containing nascent polypeptide is released from the ribosome) of these viruses due to the replacement of the 2A cs with the 3CD cs . Statistical significance was assessed by a two-way analysis of variance (ANOVA) with multiple comparisons testing. * p<0.5 ; ** p<0.01 ; *** p<0.001. Discussion Due to the constrained coding capacity of viral genomes, viruses have often evolved multiple functions within single proteins, while simultaneously exhibiting functional redundancy and overlap with other viral proteins. Moreover, they regularly function interdependently, engaging both other viral proteins and host factors to exert their role. Thus, it can be challenging to dissect the respective importance of viral factors to observed effects, and proper context truly matters. In this regard, we have been similarly constrained in our understanding of the critical security protein 2A pro . Due to the essential nature of its role in polyprotein processing, we have so far relied mostly on in vitro work, transient overexpression experiments, and surrogate infection models to study the functions of 2A pro . Although these gave key insights, they do not enable us to fully assess its function within the proper infection context. Moreover, the respective importance of the other enterovirus protease, 3C pro , towards many of the observed changes in the cell during infection, has remained convoluted. Here, we constructed viable CVB3 viruses with catalytically inactive 2A pro . We demonstrate that CVB3-2Amut viruses are hampered in their replication to various degrees in different cell lines and that these viruses are severely attenuated in a mouse model. We establish a critical role for 2A pro , and not 3C pro , during CVB3 infection in the rapid induction of NCTD via the cleavage of nucleoporins, mediating a selective translation shutdown by the cleavage of eIF4GI and PABP, suppressing SG formation, and cleaving RLR pathway proteins, inhibiting IRF3 activation and suppressing IFN-α/β gene transcription. Furthermore, we demonstrate that 2A pro is critical for overcoming early restriction by ISGs. Finally, making use of an advanced single-molecule live cell imaging approach, we reveal that 2A pro is important for the initial round of replication. In conclusion, we establish with a wide array of techniques and approaches that during CVB3 infection 2A pro , in contrast to 3C pro , plays a critical role in subverting antiviral responses and in establishing a favorable host environment to expedite viral replication. As such, 2A pro constitutes a true “security protein”; highly important for virulence and fitness, but, at least in cell culture, dispensable for viral growth 13 , 65 . Our results raise questions about the proposed roles of 3C pro in the processes described. Our data support previous suggestions that cleavage of Nup98 by 2A pro , which occurs early already upon translation of the incoming viral RNA genome and is exclusively mediated by 2A pro , is essential for triggering the NCTD. Cleavage of other nucleoporins by 3C pro late in infection may have some subtle contributions to the disruption of nuclear pore integrity in infected cells, but the relevance hereof for the viral life cycle is unknown. Furthermore, our data show that eIF4G is rapidly and exclusively cleaved by 2A pro early in infection, leading to inhibition of cap-dependent host mRNA translation. PABP, is also cleaved during infection, reportedly by both PV 2A pro and 3C pro 66 – 68 . During CVB3-2Amut infection however, no reduction in PABP levels was observed, although it cannot be excluded that 3C pro cleaves a small subset of cellular ribosome-associated PABP specifically, as suggested previously 67 . The lack of efficient PABP cleavage by 3C pro may be explained by the comparatively low efficiency of total PABP cleavage by 3C pro observed in vitro 66 . Overall, our data thus suggest that 2A pro is exclusively involved in shutting off host mRNA translation by cleaving eIF4G and PABP. Our results suggest that CVB3 2A pro is responsible for suppression of canonical SGs later in infection, in line with results obtained for EV-A71 2A pro 12 . The viral disruption of late SGs was originally attributed to the ability of 3C pro to cleave G3BP1, as mutation of the 3C pro cleavage site in G3BP1 resulted in a reduced ability to disrupt SGs, and a decrease in PV replication 48 . Strikingly, we observed fulminant SG formation, despite substantial G3BP1 cleavage during CVB3-2Amut infection. Possibly, the reduced ability to disrupt SGs in cells expressing 3C pro cleavage-resistant-G3BP1 was due to reduced replication 48 . It remains unclear how 2A pro inhibits SG formation. Yang and colleagues demonstrated that 2A pro abrogates an RNA-dependent interaction between G3BP1 and eIF4GI, which is critical to SG formation 50 . However, it is challenging to separate cause from consequence, as the observed interaction might be consequential rather than the driving factor behind SG formation. Interestingly, the L protein of EMCV, which similarly suppresses formation of canonical SGs late in infection, also abrogated the interaction between eIF4GI and G3BP1, although this protein is not a protease and thus cannot cleave eIF4GI 50 . A detailed understanding of the mechanism underlying SG inhibition by 2A pro remains to be established. Enteroviruses efficiently suppress IRF3 activation to prevent IFN-α/β gene transcription. Cleavage of both RLRs and MAVS by 2A pro and 3C pro has been implicated in this inhibition. A role of 3C pro in cleaving MAVS was proposed based on ectopic overexpression of this protease 60 . Other studies, however, showed a prominent role of 2A pro , but not 3C pro , in MAVS cleavage 61 , 62 . Consistently, we observed 3C pro cleavage products of MAVS in CVB3-infected cells only at very late time points (>12 hpi), whereas the main MAVS cleavage, perpetrated by 2A pro , was observed much earlier (>6 hpi), simultaneous with cleavage of MDA5 by 2A pro . Notably, initial transcriptional activation of IFN in CVB3-2Amut infected cells starts already at the same time point, which implies that the sensing of dsRNA has occurred before any substantial cleavage of MDA5, MAVS, or RIG-I. Granted that our ability to detect cleavage events might not be perfect, this still begs the question of how, temporally speaking, 2A pro -mediated cleavage of MDA5 and MAVS can counteract IFN gene transcription so efficiently. Possibly, other 2A pro -dependent mechanisms, such as the NCTD, contribute to this inhibition. SGs have previously been shown to serve as platforms for RLR signaling 47 , 49 , 69 , but knockout of both G3BP1 and -2, which completely abrogates the ability of Hela cells to form SGs, resulted in only a minor reduction in IFN production during EMCV-L(Zn) infection. Hence, the 2A pro -mediated inhibition of SGs fails to account for the full extent of IFN suppression. EMCV-wt efficiently suppresses IRF3 activation and IFN gene transcription through its L protein, even though RLRs and MAVS are not cleaved. Thus far, the only known activity of L is to disturb nucleocytoplasmic trafficking by recruiting ribosomal s6 kinases to the nuclear envelope where they cause hyperphosphorylation of FG-nucleoporins, such as Nup98 6 . Although the correlation between the NCTD and the suppression of IRF3 activation is poorly understood, it raises the possibility that, by analogy, the activity of 2A pro to cleave Nup98 may underly its ability to rapidly block activation of IRF3. Further research is needed to fully unravel the mechanistic underpinnings of interferon inhibition by 2A pro during enterovirus infection. The construction and characterization of CVB3 with catalytically inactive 2A pro also revealed important novel clues regarding the role of 2A pro in several other processes during CVB3 infection. For instance, we observed that CVB3-2Amut infected cells retained their regular morphology much longer, in line with development of a clear CPE only at very late time points. This might be partly explained by the lack of 2A pro -mediated cleavage of cytoskeleton proteins. Strikingly, we observed much less nuclear deformation – a hallmark of enterovirus infection - but were able to detect fragmentation of nuclear DNA. It is tempting to speculate that this might relate to faulty trafficking and mislocalization of the main DNA fragmentation factor (DFFB) due to the NCTD during CVB3-2Awt infection 70 . Furthermore, we found evidence for a function of 2A pro to modulate PKR activation. Both PKR and eIF2α become phosphorylated during CVB3 infection. Remarkably, despite the general delay in replication observed for CVB3-2Amut, phosphorylation of PKR and eIF2α seems to manifest itself even earlier than during infection with CVB3-2Awt. This implies that CVB3 2A pro proteolytic activity delays the activation of PKR and downstream eIF2α phosphorylation, in line with a previous observation with EV-A71 12 . The importance of this function remains to be established as enteroviral IRES-mediated translation has been shown to be relatively insensitive to eIF2α phosphorylation 37 , 38 . It is noteworthy that we did not find evidence for a role of 2A pro in modulating PKR and eIF2α phosphorylation when expressed as a heterologous protein in a surrogate EMCV infection model 47 , emphasizing the importance of the proper infection context to gain insight into the function(s) of viral proteins. To gain more insight into the role of 2A pro during the earliest phases of infection, we resorted to an advanced single-molecule imaging approach called VIRIM. We show that the efficiency of replication during early CVB3-2Amut infection is markedly lower, illustrated by the slower increase in the number of translating vRNA as infection progresses. Moreover, we show that in the absence of 2A pro activity, viral translation efficiency, as measured by VIRIM spot intensity, does not increase as infection progresses. It is conceivable that the lack of accessibility to proviral host factors, which normally relocalize from the nucleus upon the induction of NCTD to promote translation and replication, combined with the absence of cleaved eIF4G and other host factors thought to promote viral translation, leads to reduced efficiency in translation and replication. Distinct from the effect on the efficiency of replication in late-stage infection, we also show that the initial replication of the incoming vRNA is more vulnerable in the absence of 2A pro , as is evidenced by the higher phase 2 arrest rate. Importantly, this is unlikely due to a decrease in the amount of viral proteins that are synthesized and available for initial replication, as the efficiency of translation on the incoming vRNA during the preceding phase 1 is similar for CVB3-2Awt and -2Amut. Rather, the higher rate of failure for the first round of replication might relate to the inaccessibility of replication factors that are normally relocated from nucleus. Alternatively, it might be due to decreased RNA stability as Kempf and Barton have shown that 2A pro catalytic activity was important for viral RNA stability and to suppress host mRNA turnover machinery 71 . We previously showed that replication of incoming vRNAs is a key point at which ISGs act to restrict viral infection 14 . Here, we show that 2A pro is important to overcome the antiviral state brought about by ISGs, in line with previous work 64 , although the exact mechanism remains unclear. It remains to be established whether there is overlap in the identity of the putative restriction factors that are involved in the increased phase 2 arrest rate observed for CVB3-2Amut and the IFN-stimulated restriction factors that interfere with vRNA replication. Variations in the steady-state expression levels of such restriction factors among different cell lines may well explain the major differences that we observed in their susceptibility for efficient infection with CVB3-2Amut. Limitations Here, we thoroughly examined the respective contributions of the viral proteases 2A pro and 3C pro , within the proper infection context, to many of the processes underlying effective enterovirus replication and virulence. Notwithstanding that, our study has certain limitations, particularly as it relates to the relative importance of 3C pro . First, although we directly study many of the functions of 2A pro during infection, most of our conclusions relating to putative roles of 3C pro are based on indirect evidence. Ideally, one would similarly engineer enteroviruses with proteolytically-inactive 3C pro , but this is precluded by the sheer number of autoproteolytic cleavage events for which 3C pro is essential during polyprotein processing. Second, we do not find a clear role of 3C pro voor certain processes in which it was previously implicated. One might argue that the absence of such effects could relate to the relatively low expression level of 3C pro during CVB3-2Amut infection due to less efficient replication. However, the observation of other 3C pro cleavage events, such as those of PKR, G3BP1, and RIG-I, argues against such a notion. Third, we mostly made use of HeLa cells as a model system, because CVB3-2Amut replicated relatively well in this cell line, giving us a good window for assessing the role of 2A pro in many processes. In other cell lines, such as A549, CVB3-2Amut replication was much more compromised, the reason for which is unknown. Possibly, this might relate to higher steady-state expression of specific restriction factors that need to be countered by 2A pro , but this requires further study. Finally, our study on the importance of 2A pro was restricted on CVB3, which is a commonly used model enterovirus. However, we argue that – based on the evolutionary conserved functions of the enterovirus proteases 47 , 72 – our results have broad implications for a better understanding of the interplay of other enteroviruses with host cells as well. Materials & methods Cell lines HEK293-T (ATCC CRL-3216), HeLa-R19 (RRID: CVCL_M763), BGM (RRID: CVCL_4125), Vero E6 (ATCC CRL-1586), A549 (ATCC CCL-185), Hela-R19 saGFP-STAb BFP- NLS STAb (derived from Hela-R19, made as described previously in Boersma 2020, Bruurs 2025) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (V/V) fetal bovine serum (FBS) and 100 units/ml penicillin and streptomycin (P/S). Virus design and production Recombinant CVB3 viruses were derived from the pRibCVB3/T7 plasmid, which contains the cDNA of CVB3 strain Nancy driven by a T7 RNA polymerase promoter (Wessels et al., 2006 73 ). Mutations in the 2A pro sequence (H21A, D39A, and C110A) were introduced by site-directed mutagenesis of the pRib infectious clone. The 2A pro cleavage site between the P1-P2 region was replaced by: (I) the IRES sequence of Theiler’s encephalomyelitis virus (TMEV) genome, (II) a self-cleaving peptide from Thosea asigna virus (T2A), or (III) a sequence corresponding to the 3C/3D cleavage site of CVB3 recognized by 3C pro (3CD cs ). 5x-Suntag-CVB3 viruses were generated as described earlier (Boersma et al., 2020 14 ). Briefly, we introduced a 5x-SunTag cassette with a 3CD cleavage site upstream of the VP4 region of the main viral ORF in the infectious cDNA clone plasmids of CVB3-wt, CVB3-3CDcs-2Awt, or CVB3-3CDcs-2Amut. Renilla luciferase (RLuc)-CVB3 viruses were generated by introducing the Renilla luciferase gene upstream of the VP4 gene separated by the 3C/3D cleavage site recognized by 3C pro . Virus stocks were generated by transfecting purified in vitro transcribed viral RNA (RiboMAX, Promega) in HEK293-T cells. After 2-4 days, when total cytopathic effect (CPE) was visible, the supernatant containing virus was subjected to three freeze-thaw cycles, cell debris was pelleted at 4,000x g for 15 minutes and the virus was concentrated from the supernatants by ultracentrifugation through a 30% sucrose cushion at 140,000x g for 16 hrs at 4°C in a SW32Ti rotor. Virus pellets were resuspended in phosphate-buffer saline (PBS), aliquoted, and stored at −80°C. The viruses were characterized by isolating viral RNA from 150 μl aliquots of the cell culture supernatant with the NucleoSpin RNA Virus kit (Macherey-Nagel) followed by conventional RT-PCR and bidirectional Sanger sequence analysis of the inserted (I) IRES, (II) T2A, or (III) 3CDcs sequence between P1-P2 region, 2A pro and 5x-SunTag array sequence. Viral titers, determined on HeLa cells by endpoint titration and calculated by the Spearman-Kaerber formula, are averaged from three independent experiments. Mice experiment materials & methods SJL/JRj mice (6 weeks old) were purchased from Janvier Labs (France) and were infected intraperitoneally with equivalent viral doses (either 1.0*10 7 TCID 50 /ml or 5.0*10 5 TCID 50 /ml) of CVB3, CVB3-2Awt or CVB3-2Amut. At day 3 post-infection (pi), mice were euthanized by intraperitoneal (IP) injection of 100 μL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA) and pieces of pancreas were collected for quantification of viral RNA, infectious virus titers by RT-qPCR, and end-point virus titration, respectively. In addition, other pieces of pancreas were fixed in 4% formaldehyde for histopathological analysis. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P179/2023). Pancreatic histopatology was assessed and scored as follows. For necrosis: 0 = tissue unaffected, 1 = <10% of tissue affected, 2 = 11-25% of tissue affected, 3; 26-50% of tissue affected, 4 = more than 50% of tissue affected; for inflammation and edema: 0 = none, 1 = mild, 2 = moderate, 3 = severe. Analyses were conducted blinded to the experimental groups. For each mouse, 10 field of views obtained with a 20x objective were analyzed and the median scores determined. Growth curve and quantitative PCR (RT-qPCR) HeLa R19 cells were seeded in flat-bottom 96-well plates (10 4 cells/well) and after 16 hrs recovery were infected with the recombinant CVB3 at MOI=5 TCID 50 /ml for 30 min. At indicated time points post-infection plates were either freeze-thawed three times to lyse the cells for titration or lysed using RNA lysis buffer for isolation of total RNA (Machery-Nagel) for RT-qPCR. Reverse transcription was set up using TaqMan Reverse Transcription Reagents (ThermoFisher) and qPCR analysis was performed using specific primers for IFN-β (5’-ATGACCAACAAGTGTCTCCTCC-3’ and 5’-GCTCATGGAAAGAGCTGTAGTG-3’), IFIT1 (5’-TGCTCCAGACTATCCTTGACCT-3’ and 5’-TCTCAGAGGAGCCTGGCTAA-3’), β-actin (5’-CCTTCCTGGGCATGGAGTCCTG-3’ and 5’-GGAGCAATGATCTTGATCTTC-3’), and CVB3 viral RNA (5’-CGTGGGGCTACAATCAAGTT-3’ and 5’-TAACAGGAGCTTTGGGCATC-3’) using the Fast SYBR green Master Mix (ThermoFisher). Relative levels of IFIT1, IFN-β mRNA and vRNA were normalized to actin expression. In each experiment, expression was determined from three technical replicates. Luciferase assay HeLa R19 cells (10 4 cells/well) and HEK293-T cells (1.2×10 cells/well) were seeded in 96-well plates. After 16-hour recovery, cells were transfected with in vitro transcribed RLuc-CVB3 RNA using Lipofectamine2000 (Invitrogen). At indicated time point post infection, cells were washed with PBS and lysed in Renilla luciferase assay lysis buffer (Promega). Luciferase activity was measured using a Renilla luciferase assay system (Promega) with a GloMax Explorer luminometer (Promega). Luminescence was measured as relative light units per second (RLU/s). For the interferon (IFN) pretreatment experiment, HeLa cells were treated with the indicated concentrations of IFN-α2 (Sigma-Aldrich) for 16 hrs. Cells were infected with RLuc-CVB3 recombinant viruses at MOI of 0.1 TCID 50 /ml for 30 min. Following infection, the virus inoculum was removed and replaced with complete medium containing IFN-α2 at the same concentrations used during the pretreatment phase. Luciferase activity was measured as described above. Antibodies The following antibodies were used for WB analysis: rabbit anti-eIF4GI (1:1000, Cell Signaling, #2498), rabbit anti-DAP5 (1:1000, Invitrogen, #PA-21377), mouse anti-PABP (1:1000, SantaCruz, #sc-32318), anti-PKR (1:4000, BD Biosciences, #BDB610764), rabbit anti-PKR-p T446 (1:1000, Abcam, #ab32036), rabbit anti-eIF2α-p (1:1000, Abcam, #32157), rabbit anti-Nup98 (1:1000, Cell Signaling, #2598), rabbit anti-G3BP1 (1:1000, Aviva, #ARP37713-T100), rabbit anti-MDA5 (1:5000, kindly provided by Paola Barral), rabbit anti-RIG-I (1:500, Abgent, #AP1900a), rabbit anti-MAVS (1:2000, Enzo Life Science, #ALX-210-929), rabbit anti-IRF3 (1:1000, Abcam, #ab25950), rabbit anti-IRF3-p (1:1000, Cell Signaling, #4947), rabbit anti-PARP (1:1000, Roche, #11835238001), rabbit anti-Caspase7 (1:1000, Cell Signaling, #9492), rabbit-anti CVB3 2C (1:1000, kindly provided by Lindsay Whitten), mouse anti-β actin (1:5000, Sigma-Aldrich, #A5441r,). Respective IRdye680- or IRdye800-conjugated secondary antibodies (LiCOR) were used for detection. For immunofluorescence analysis staining procedures, the mouse anti-dsRNA (1:1000, Jena BioScience, #RNT-SCI-10010), rat anti-Nup98 (1:200, Sigma-Aldrich, #N1038), rabbit anti-hnRNPK (1:250, ThermoFisher, #PA5-19484), goat anti-eIF3η (1:200, SantaCruz, #sc-16377), rabbit anti-G3BP2 (1:200, Bethyl Laboratories, #A1302-040A), goat anti-TIA-1 (1:200, SantaCruz, #sc-1751), rabbit anti-Sam68 (1:200, SantaCruz, #sc-333), antibodies were used. Alexa Fluor 405-, Alexa Fluor 488-, Alexa Fluor 555-, Alexa Fluor 594-, Alexa Fluor 647-conjugated (1:200, Invitrogen) secondary antibodies were used for detection. Western blot analysis HeLa R19 cells were seeded in 6-well clusters (5×10 5 cells/well) and after a 16 hrs recovery cells were infected with recombinant CVB3 at MOI 10 TCID 50 /ml. At the indicated time point, cells were lysed with SDS sample buffer (Tris-HCl pH 6.8, 50mM, Glycerol 1M, Bromophenol Blue 1.5mM, DTT 100 mM, SDS 2% w/v). Samples of each lysate were separated by SDS-PAGE in reducing 8% polyacrylamide gels. The proteins were then blotted onto 0.2 µm nitrocellulose membranes by wet electrophoretic transfer. Membranes were washed three times in TBST (20 mM Tris, 150 mM NaCl + 0.1% Tween-20), 5 min each, and incubated in blocking buffer (TBST + 5% BSA) for 1h at RT. Membranes were successively incubated with primary antibodies diluted in blocking buffer for 16 hrs at 4°C, and then for 30 min at RT with secondary antibody diluted in blocking buffer. Between and after the incubations, the membranes were washed, thrice each time, with TBST. Finally, membranes were washed once with PBS and scanned using an Odyssey Imager (Li-COR). Immunofluorescence analysis HeLa R19 or HeLa-3xNLS-GFP cells were seeded onto 12 mm glass coverslips in 24-well plates (Corning Costar) at a density of 5×10 4 cells per well, grown for 24h and infected with CVB3 recombinant viruses. Virus-containing medium was incubated on the cells for 30 min at 37°C and after incubation time replaced with complete DMEM. At the indicated time post-infection, cells were fixed in PBS + 3.7% paraformaldehyde (PFA) for 30 min, incubated with PBS + 0.1% glycine for 10 min, permeabilized with 0.1% Triton X-100/PBS+S for 10 min at room temperature (RT) and blocked in blocking buffer (PBS + 1% BSA + 0.1% Tween-20) for 30 min. Cells were then incubated in blocking buffer with primary antibodies for 1h at RT. After washing the cells with PBS+0.1% Tween-20, cells were incubated with secondary antibody in blocking buffer for 1h at RT. Cells were then washed three times with PBS+0.1% Tween-20, once with distilled water, and mounted on glass microscope slides in ProLong Diamond Antifade (Invitrogen) mounting medium. Slides were examined by confocal immunofluorescence microscopy (Nikon A1R or Evident SpinSR10 IX83) with a 40x Oil immersion objective (Nikon CFI PlanFLUO NA = 1.3, or Evident UPLAPO NA = 1.4). Sequential laser acquisition was performed (405, 488, 561 and 645 nm) over quadband dichroic filter and bandpass emission filters to separate Alx405/BFP (Nikon: 482/35, Evident: 477/60), for AausFP1/GFP/Alx488 (515/30), Al×555/595 (Nikon: 595/50, Evident: 607/36) and Alx647 (Nikon: 700/75, Evident: 685/40) channels. Images were analyzed by manual scoring in a blinded fashion by two independent observers using ImageJ/FIJI software 74 using the criteria outlined below. Quantification of stress granule formation Images for SG quantification were obtained as described in immunofluorescence analysis. All Analyses were performed fully blinded to the experimental groups. For each time point and virus condition 15 40x field-of-views (FOVs) were obtained and analyzed as follows. (i) all cells within a FOV were counted based on DAPI stain. cells were excluded if they were partially outside of the FOV. (ii) All dsRNA-positive cells were marked based on anti-dsRNA J2 antibody staining. (iii) positivity for SGs was assessed based on assessment of G3BP1 and eIF3ρι signal. SGs were either scored as large or small based on size, and upon the observation that the small early SGs were always negative for eIF3ρι, whereas the large SGs were always positive. Moreover, cells with small early SGs were mostly negative for dsRNA. Live cell imaging and VIRIM Hela-R19 AausFP1-STAb (a.k.a. saGFP-STAb) BFP-NLS Clone-1 cells were seeded at 1.5×10 5 cells per well in a 18-well coverglass-bottom imaging chamber (Ibidi, cat#: 81817) and incubated overnight at 37°C. Cells were infected with indicated 5x-SunTag viruses at MOI of 0.25 in DMEM (10% FCS, 1% P/S) for 30 min at 37°C. The inoculum was removed and replaced with Leibovitz’s L15 medium without phenol red (ThermoFisher Scientific, cat#: 21083027) supplemented with 10% FCS and 1% PS. Cells were then imaged on an Evident SpinSR10 IX83 SoRa spinning disc confocal microscope (UPLAPO60xOHR NA = 1.5 or UPLXAPO40xO NA = 1.4 objective, Yokogawa CSU-W1 SoRa, Hamamatsu sCMOS ORCA-Fusion C14440) equipped with a temperature- and CO 2 -controlled imaging chamber (CellVivo, Evident) using cellSens Dimension software (Evident, vs4.1). Time-lapse images were recorded overnight at 3 min interval with 15 Z-slices of 0.7 μm stepsize, covering the entire cell volume. Several positions were selected randomly throughout the well(s) of interest. Image processing and VIRIM analysis Before all time-lapse analyses, a maximum intensity projection was performed using Evident CellSens software, and the projections were used for all further analyses. For all intensity analyses, a bleach correction was performed using ImageJ software using the “exponential fit” model. All following image analysis was performed using ImageJ software and recorded/processed in Microsoft Excel and GraphPad PRISM. For all VIRIM analyses, at least 3 independent datasets were obtained and analyzed. VIRIM analysis and quantification All VIRIM analyses were done as previously described 14 , 75 . Briefly, VIRIM translation spots were readily distinguishable from background spots and aggregates on basis of mobility, size, and appearance. Each position was screened for analyzable cells based on the following set of inclusion and exclusion criteria. Cells were excluded from analysis if: (i) they had a translation spot in the first timeframe, (ii) they were not entirely within the field-of-view during the entirety of analysis, (iii) they underwent cellular division during analysis, (iv) the first translation spot appeared clearly in a second round of infection (after > 6h), (v) the imaging could not be followed up until phase 5 or a clear phase 2 arrest, (vi) they were double infections (i.e., more than one spot appears in phase 1). Definition of VIRIM phases Phases were called upon observation of at least 4 consecutive frames in which a spot was present or absent. Phase 1, translation of the incoming vRNA, was defined as the initial appearance of a single VIRIM translation spot. After a while, this initial spot disappears, marking the start of phase 2, the first round of replication. Next, one of three things can happen; (i) there is a reappearance of multiple new translation spots, marking successful replication and the start of phase 3, (ii) there is no reappearance of any spots, signifying replication failure and infection arrest, or (iii) there is reappearance of a single spot, signaling a replication failure, and recommencement of translation on the initial incoming vRNA. The latter scenario can occur in a recurring pattern of translation followed by replication, which are called translation pulses and breaks, which finally results in either successful start of phase 3 or phase 2 arrest. Here, all translation breaks and pulses up until the final replication break before success or failure were collectively called phase 1. Phase 3 constitutes the initiation of translation on nascent vRNAs synthesized during initial replication, and is marked by a steep increase in the number of translation spots. Once this stabilizes at a constant number, phase 4 is called, which is manifested by continued translation and a new round of replication. The end of phase 4 is marked by a second steep increase in the number of VIRIM translation sites, and this is called phase 5. Once the number of spots exceeds 50, we are no longer able to confidently distinguish between individual spots, marking the end of our analysis. Quantification of VIRIM translation site intensity To identify the intensity of VIRIM translation sites we measured the mean intensity in a square region of interest (ROI) of 8×8 pixels centered on the translation spot. To correct for background and STAb expression levels, we subtracted the average intensity of three randomly selected 8×8 pixel ROIs on surrounding cytosol of the same cell. If spots appeared very close together, or were spherical (indicating this is likely the same spot moving between multiple Z-frames), they were excluded from analysis. For phase 1 spot intensity we measured the spot intensity up to the first 10 frames (with at least 5, else it was excluded from analysis), and averaged this. For phase 4, the first two or three frames were measured, with maximum 5 spots per frame, for a total of 10 spots. If more than 5 spots were present in one frame, spots were selected starting from the upper-left side of the cell, and working to the right and down, until 5 frames were measured. The lower VIRIM spot intensity we observed for CVB3-wt compared to CVB3-3CDcs-2Awt can be explained by the replacement of the 2A pro with the 3CD pro cleavage site at the P1-P2 junction, as this means that the nascent polypeptide chain, and consequently the associated STAb-GFP, will stay attached to the translating ribosome for a longer time ( Fig. 8b , Supplemental Fig. 5b ). Quantification of nuclear leakage of NLS-BFP Only those infections where at least 10 frames prior to the start of phase 1 could be analyzed were selected. Measurements were done starting 10 frames prior to the start of phase 1, or in uninfected cells, at the start of the time-lapse movie. For each frame, three 5×5 ROIs were randomly selected in the cytosol, and 3 5×5 ROIs were randomly selected in the nucleus (avoiding the nucleoli). Both the edges of the cell, as well as the edges of the nucleus were excluded. The cytosol also contained areas where signal was consistently less than in surrounding cytosol, likely representing organelles. These were also excluded. The BFP intensity was measured for each frame, and the three ROIs were averaged. The mean cytoplasmic BFP intensity was substracted in each frame from the nuclear BFP intensity and this was normalized to the average of the 3 frames preceeding appearance of the first spot OR the first 3 frames in a movie if uninfected. In vivo metabolic labelling Newly synthesized proteins were determined by metabolic labeling using a threonine-derived non-canonical amino acid β-ethynylserine (βES), followed by fluorescence labeling of βES based on an adapted protocol of the published THRONCAT assay 36 . In particular, HeLa R19 cells were seeded in 6-well plates (4.0×10 5 cells/well) and after a 16 hrs recovery they were infected with recombinant CVB3 at MOI 100 TCID 50 /ml. After 1h, viral inoculum was replaced with complete DMEM. One hour prior to cell lysis, cells were incubated with 2µM (βES) in complete DMEM at 37°C for 1h and either left untreated or treated with 500 µM sodium arsenite (NaAsO2, Riedel-de Haen) to generate a positive control for translation arrest. Following incubation, at the indicated time point, cells were washed with PBS, lysed with lysis buffer (HEPES 50 mM pH 7.4 (Sigma-Aldrich), NaCl 150 mM (ThermoFisher), Triton-X-100 1% (Sigma-Aldrich), 1 × EDTA-free protease inhibitor cocktail (Roche)), and subjected to sonication on ice. Cell lysates were cleared by centrifugation at 4°C, after which the supernatant was transferred to a new tube and the protein concentration was determined by BCA assay (Micro BCA Protein Assay kit, ThermoFisher). Lysates were flash-frozen and stored at −80°C until further usage. Cy5 conjugation to HeLa proteome via azide-alkyne click chemistry was performed as described by Ignacio et al., 2023 36 . Protein samples were run on an SDS-PAGE and Image Quant800 Imager (Cytiva Amersham) was used for image acquisition. Statistical analysis Data display and analysis were performed using GraphPad Prism version 10 (GraphPad Software, CA). Appropriate statistical analyses were performed as indicated using GraphPad Prism 10. Unless otherwise indicated, all statistical tests assumed normal distribution of data and were done using an α-value of 0.05 as cutoff for statistical significance. Software Figures were made in Adobe Illustrator 2023. Most of the schematics were produced using BioRender.com . Data availability Raw data is available via Mendeley Data using link: LINK. Due to size limitations, for the time lapse imaging data representative raw pictures will be made available for download. The entire dataset can be accessed upon request with the corresponding author. Author contributions J.G.S, C.A. and F.J.M.v.K. conceptualized the project; J.G.S, C.A., S.O.S., R.A., J.M.A.v.d.B, L.J.M.B., H.J.T., J.N., M.E.T. and F.J.M.v.K. developed the methodology; J.G.S, C.A., S.O.S., M.Z., A.L.W.v.V and R.A. performed the experimental work; J.G.S, C.A., S.O.S., M.Z., R.A., D.R. and J.M.A.v.d.B conducted formal analyses; J.G.S., C.A., S.O.S, M.E.T. and F.J.M.v.K curated the data; B.I. and K.M.B. developed and provided reagents; R.W.W. and L.J.M.B. provided support and advice with live cell imaging; J.G.S., C.A., S.O.S and F.J.M.v.K wrote the original manuscript, which was subsequently reviewed and improved by all authors; H.J.T, J.N., M.E.T. and F.J.M.v.K supervised the work; S.O.S., J.N., M.E.T. and F.J.M.v.K acquired funding. Competing interests The authors declare no competing interests. Supplementary figures Download figure Open in new tab Supplementary Fig. 1: Growth kinetics of 2Awt and 2Amut viruses on different cell lines. ( a ) CVB3, 3CD cs -2Awt, and 3CD cs -2Amut viral stocks were titrated on HeLa-R19, BGM, Vero E6 and A549 by end-point dilution. ( b ) Growth curves of CVB3, T2A-2Awt and T2A-2Amut viruses in HeLa-R19, BGM, Vero E6 and A549 cells performed as in Fig.1e. Data represent the mean ± SEM of the three technical replicates ( a , b ). Download figure Open in new tab Supplementary Fig. 2: Cleavage of PABP at later time points and SGs in infected cells stained with TIA-1 and Sam68. ( a ) Western blot analysis for PABP. Cell lysates derive from the same experiment shown in Fig. 4d . ( b ) IF analysis of HeLa-R19 cells infected with CVB3, 3CD cs -2Awt and 3CD cs -2Amut viruses, fixed and stained for dsRNA as infection marker and TIA-1 and Sam68 as SG marker. Experiment was performed as described in Fig. 4c . Magnified regions are shown in white boxes. Scale bar: 25 μm. Download figure Open in new tab Supplementary Fig. 3: MAVS cleavage products generated by 3C pro and 2A pro in CVB3-2Awt and CVB3-2Amut infected cells. Western-blot analysis of MAVS in cell lysates obtained from the same experiment shown in Fig. 4d . Black triangles denote intact proteins, white triangles the cleavage products. Download figure Open in new tab Supplementary Fig. 4: 2A pro rapidly and exclusively triggers nucleocytoplasmic trafficking disorder. Hela-R19 saGFP-STAb BFP-NLS C1 cells were infected at an MOI of 0.25 with ST-CVB3-2Awt or ST-CVB3-2Amut and time-lapse imaging of STAb-GFP and BFP-NLS was performed. ( a ) Representative pictures of cells infected with ST-CVB3-2Awt or -2Amut at different time points aligned to the start of phase 1. ( b ) average normalized BFP-NLS intensity ratio between nucleus and cytoplasm over time aligned to start phase 1. Scale bar: 10 μm. ( c ) Kaplan-Meier curves depicting duration of phase 1 in those cells with successful replication, non-successful replication (i.e., phase 2 arrested), or both (total). ( d ) Fraction of infections with indicated number of translation pulses. Download figure Open in new tab Supplementary Fig. 5: VIRIM spot intensity as a measure of viral translation efficiency. ( a ) Schematic representation of VIRIM spot intensity analysis. Upper panel shows a situation with comparatively lower translation efficiency, less translating ribosomes on a single vRNA, and consequently a smaller accumulation of fluorescence signal. The lower panel depicts the scenario of a comparatively higher translation efficiency. ( b ) Absolute intensity measurements of different CVB3 mutants. The higher intensity for 3CDcs-2Awt is the consequence of a relatively longer effective transcript length (i.e., the length of transcript that is decoded before the nascent peptide chain is released from the ribosome). statistical significance was assessed by a one-way analysis of variance (ANOVA) with multiple comparisons testing. * p<0.5 ; ** p<0.01 ; *** p<0.001. Acknowledgements & Funding We thank all members of the Virology section at the Faculty of Veterinary Medicine of Utrecht University for helpful discussions. This work was financially supported by NWO Klein-2 Grant (OCENW.KLEIN.344) to M.E.T. and F.J.M.v.K; European Commission (EC) Marie Sklodowska-Curie Actions (MSCA) Innovative Training Network H2020-MCSA-ITN-2019 (INITIATE, Grant Nr. 813343) to F.J.M.v.K; EC ERC Advanced Grant to F.J.M.v.K (virLUMINOus, Grant Nr. 101053576); EC ERC Consolidator Grant (VirIm, Grant Nr. 101044794) to M.E.T; the Belgian Federal Government for the VirusBank Platform to J.N.; and EC MSCA Postdoctoral Fellowship (BARONET, Grant Nr. 101152667) to S.O.S. Furthermore, M.E.T. was financially supported by the Oncode Institute, which is funded in part by the Dutch Cancer Society (KWF). Footnotes ↵ * These authors share senior authorship. References 1. ↵ Family: Picornaviridae | ICTV. https://ictv.global/report/chapter/picornaviridae/picornaviridae . 2. ↵ Chen , Y. G. & Hur , S . Cellular origins of dsRNA, their recognition and consequences . Nat Rev Mol Cell Biol 23 , 286 – 301 ( 2022 ). 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