Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption

Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption | 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 Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Afagh Bapirzadeh, MITRA Salehi, ZARRIN Minuchehr, Bijan Bambai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7546879/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract NSP1 protein is one of the first produced proteins of SARS-CoV-2, the virus which is responsible for Corona disease, sited at the beginning of 5 ́ end of virus gene number one. NSP1 bonds to Human 40S ribosomal subunits has potential roles in host cells mRNA translation inhibition.to clarify subtle molecular reasons of carrier animal’s pathogenicity, we used bioinformatics tools and 7k5i file structure acquired from PDB data bank to investigate interactions and internal bonds of 40S ribosomes in normal cell conditions. NSP1 protein bonds in complex with ribosomes of Human host cells in individuals affected by SARS-CoV-2 differ from normal states. Majority of NSP1 interactions involve 18SrRNA ribosomal subunit and one of ribosomal proteins. These bonds are at A 605 and G 600,601 nucleotide positions in 18SrRNA leading to both physically and spatially disrupting existing internal interactions in this subunit which eventually could cause ribosome to lose its function. Additional to 18SrRNA, NSP1 also bonds with three ribosomal proteins: S2, S3 and S30.alignment comparison of these molecules in Human and other mammalian in one hand and between Human and carrier animals such as camel and Manis in another hand showed for example camel S2 protein is more like Manis instead of being genetically close to human or cows. These crucial findings strongly support important role of NSP1 in translation inhibition of host cells ribosomes.by using these results and making a few structural changes at carboxyl end of NSP1 protein suppression and restriction of cancerous cell growth can be gained. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Microbiology Biological sciences/Molecular biology SARS-CoV-2 NSP1 Bioinformatics 40s ribosomal subunits Bats Manis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Points NSP1 interactions in human 40S ribosomal complex and involved proteins Comparison between human ribosomal sequences and protein sequences and intermediate animals Introduction According to the World Health Organization (WHO) statement in March 2020, the rapid global spread of coronavirus disease (COVID-19) led to its declaration as a global pandemic ( 1 ). In response, the Health Ministry of the Islamic Republic of Iran, together with the government, announced a national quarantine and established a specialized committee tasked with implementing social education and prevention strategies. Among the various measures, authorities encouraged researchers and provided funding for projects focused on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19. Notably, the Iran National Institute of Genetic Engineering and Biotechnology has mobilized several research groups to investigate different aspects of this disease( 2 ). COVID-19 is initiated when the SARS-CoV-2 virus enters human host cells via the binding of its structural spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface( 3 ). The viral genome is composed of a single positive-sense RNA that encodes 11 open reading frames (ORFs). Following transcription and translation, these ORFs yield several polyproteins that are essential for viral replication and survival. These polyproteins are subsequently processed by viral proteases, including the main protease (Mpro), the papain-like protease (PLP), and the 3C-like protease (3CLpro). One of the resulting proteins, non-structural protein 1 (NSP1), is a 20 kDa factor localized in the cytoplasm of infected cells( 4 , 5 ). Yoshimoto has suggested that NSP1, also referred to as the leader protein, plays a crucial role in inhibiting host cell gene expression, likely by binding to the 40S ribosomal subunits( 6 ). This protein, which is also called the host shutoff factor, suppresses host innate immune functions( 7 ). Ribosomes are essential cellular organelles composed of protein and RNA subunits that are responsible for protein synthesis. For NSP1 to exert its function, it must bind to the 40S ribosomal subunit ( 8 ). This binding is thought to influence the ribosome’s architecture by disrupting the intrinsic interactions among its proteins and RNA( 9 ). We hypothesize that when NSP1 engages with ribosomal subunit components, it perturbs both internal and external contacts—potentially causing contact inhibition between adjacent nucleotides or proximate amino acids—and thereby impairs normal ribosomal function. According to Schubert and colleagues, the full-length 5′ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes( 7 ). The novel coronavirus is transmitted to humans via potential intermediate hosts. Reservoir species, such as horseshoe bats, have experienced multiple recombination events among different SARS-related coronavirus ancestors ( 10 , 11 ). Other animal species, including pangolins and masked palm civets , have also been proposed as potential intermediaries in the transmission chain( 12 ). In addition, studies conducted in New Zealand on rabbits , ferrets , and hamsters have shown that although these animals can harbor the virus asymptomatically, they do not serve as true reservoirs( 13 , 14 ). It is noteworthy that these potential intermediate hosts, even when in contact with the virus, are neither diagnosed as infected nor do they exhibit clinical symptoms( 13 , 14 , 15 ). In this article, we demonstrate that NSP1 interactions with the human 40S ribosomal complex differ markedly from those observed in potential intermediate hosts. In these animals, modifications such as deletions, insertions, or other sequence variations in ribosomal proteins result in altered binding interfaces with the viral NSP1. Such changes likely lead to weaker or even absent interactions, which may contribute to their status as carriers without manifesting disease. To examine this hypothesis, we first analyzed the structure of NSP1 and detailed its interactions with the 40S human ribosome, identifying critical hotspot sites. Subsequently, we compared these interaction regions between humans and potential intermediate hosts Method In December 2020, Shi and colleagues determined the crystallographic structure of the SARS-CoV-2 NSP1 protein in complex with the 40S ribosome( 16 ). This structure, available from the Protein Data Bank under the identifier 7K5i, forms the fundamental basis of our study(. We utilized the free educational version of PyMOL (version 2.4.1) to examine the binding interface between NSP1 and the human 40S ribosomal complex. Specifically, we investigated interactions between NSP1 and four key components: the 18S rRNA and the ribosomal proteins S30, S3, and S2 (see Table 1). For detection of contacts, we applied a distance cutoff of 4 Å to define both polar (e.g., hydrogen bonds) and non-polar interactions using PyMOL’s measurement tools and built-in interaction analysis features. Table 1. NSP1 interactions with 40S human ribosome (18SrRNA and S2, S3, S30). polar contacts are shown with star Given the multiple interactions between NSP1 and the human ribosome, we extended our analysis to compare the relevant ribosomal sequences between humans and several potential intermediate host species(17,18,19). The species examined include bats (genus Rhinolophus and Myotis ), pangolins ( Manis pentadactyla and Manis javanica ), the American pika ( Ochotona princeps ), and camels ( Camelus dromedarius and Camelus ferus ). In addition, species with close evolutionary relationships to humans—such as the mouse ( Mus musculus ), cow ( Bos taurus ), monkeys (genera Mandrillus and Saimiri ), gorilla ( Gorilla gorilla ), chimpanzee ( Pan troglodytes ), and sheep ( Ovis aries )were included in the sequence alignments(20). To perform these alignments, we initially retrieved the NSP1-interacting protein and ribosomal RNA sequences from the National Center for Biotechnology Information (NCBI) database. Multiple sequence alignments were then executed using Clustal Omega, a freely available, non-commercial web tool. The results of these alignments, which served to highlight differences in the putative NSP1 interaction sites between humans and the examined species, are summarized in Tables 2, 3, 4, and 5. It is important to note that in some cases data availability was limited due to incomplete protein or genome sequences in public databases.This integrated methodological approach enabled us to systematically compare the interaction interfaces of NSP1 with 40S ribosomal components across species. The insights garnered from these analyses provide a potential explanation for the differential susceptibility to SARS-CoV-2 infection observed between humans and various intermediate hosts. Results In table 1 the most polar interactions are between NSP1 protein and 18SRNA. 18SRNA is the biggest subunit of 40S ribosome. The interactions between 18SRNA and NSP1 are not only polar but also consist of four non-polar bonds under 4 angstroms (A°). NSP1 at Lys-164, His-165, Ser-167, Arg-171, Arg-175 amino acids have polar contact with 18SRNA nucleotide bases. Among this Arg-175 from NSP1 has polar bonds with A 605 and G 606 , Arg-171 with G 600,601 and U 607 . Lys-164, His-165 and Ser-167 have both polar and non-polar bonds with 18SRNA nucleotides. Ser-167 has a polar contact with G 600 but a non-polar contact with G 601 . His-165 also has polar bond with U 630 and a non-polar bond with U 607 . In Lys-164 case as it can be seen in Table 1, there are two polar bonds with G 625 and C 624 and one non-polar bond with U 631 . Also Glu-172 has one non-polar bond with U 607 base of 18SRNA. (see Fig. 1 ) Among proteins involved in ribosomal translation system helping 40S subunits and interacting NSP1, there were three specific protein S2, S3 and S30. S30 has the lowest connection to NSP1, one polar contact between Lys 52 from S30 protein with Glu-176 and a non-polar contact between Lys 52 and Arg-175 from NSP1 protein (see Fig. 2 ). The last protein which will be discussed is S2 from 40S ribosomal complex. NSP1 in Asn-178 and Gln-158 form polar contacts with Val 106 and Val 147 , respectively. These two polar bonds were the only polar contacts between NSP1 and S2, other contacts were all non-polar. Gln-158 in three positions of Glu 146 , Val 147 and Phe 124 had non-polar contacts. Trp-161 from NSP1 had non-polar connections to Phe 124 , Thr 122 and Pro 111 . Phe-157 also had non-polar connections with Phe 124 and Ile 109 from S2 protein. In addition to Asn-178, Met-174 also had one non-polar bond with Val 106 from NSP1. (see Fig. 4 ) The next step in finding NSP1 interactions with ribosomal complex was alignment among human 18SrRNA and its dependent proteins with intermediate animals. One of the most significant differences in attachment sites of NSP1 with human ribosomes compared to camel, bats and pangolins specifically were seen in S2 protein. Camel S2 protein sequence ( Camelus dromedaries ) which is shown in Table 2, had a relatively huge deletion, starting from amino acid number 48 and lasts to amino acid number 80. In between this deleted segment, there were two important amino acids, Glu-146 and Val-147 which were NSP1 attachment points to human S2 ribosome. This deletion doesn’t exist in similar sequences of human ribosomal protein S2.Another two differences come from camel S2 protein amino acids 34 and 37 in which instead of Arg and Phe, Glu and Leu were replaced. It is important to mention that camel is one of Corona virus intermediate animals who don’t get infected by the virus and is only a carrier. S2 protein of isoform X1. a type of monkey ( Mandrillus leucophaeus ) also had Threonine instead of Isoleucine in the position of 109. (see Table 2) Table 2. ribosomal S2 sequence alignment Human S2 ribosomal protein LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Camelus dromedarius LKDE V LK I M P VQKQTRAGQ QT R L KAFVATGDDKG---------- -- ----- Camelus bactrianus LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Mus musculus LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Bos taurus LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Manis pentadactyla LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Manis javanica LKDD V LK I M P VQKQTRAGQR T R F KAFVAIGHYNGHVGLGX—SK EV ATAIR Myotis brandtii LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Ochotona princeps LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Gorilla gorilla LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Mandrillus leucophaeus isoform X1 LKDE V LK I M T VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Mandrillus leucophaeus LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Pan paniscus LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Rhinolophus ferrumequinm LKDE V LK I M P VQKQTRAGQR T R F KAFVAIGDYNGHVGLGVKCSK EV ATAIR Saimiri boliviensis LKDE V LK I M P VQKQTRAGQR T R F KEFVAIGDYNGHVGLGVKCSK EV ATAIR Ovis aries LKDE V LK I M P VQKQTRAGQR T R F KAFVAIADYNGHVGLGVKCSK EV ATAIR S3 ribosomal protein similar to S2 protein showed differences between human and intermediate animals. For example, a 126 amino acids deleted segment at the beginning of this protein in X2 and X3 isoforms of Manis pentadactyla and Manis javanica existed. This deletion was responsible for protein size differences. Aside from this fact, five attachment points in NSP1 protein including Leu-113, Ala-114, Val-115, Arg-116 and Arg-117 were also deleted (see table 3). Sequence Alignments between 18SrRNA of human 40S ribosomal and intermediate animals showed no significant differences and all attachment points to NSP1 existed for all of them (see table 4). This also applied for S30 protein. Having only 59 amino acids length in human S30 protein in contrast with longer length of this protein in intermediate animals was a very important fact (see table 5). Table 3. ribosomal S3 sequence alignment Human S3 ribosomal protein QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Camelus dromedarius QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Mus musculus QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Bos taurus QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Manis pentadactyla isoform X1 QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Manis pentadactyla isoform X2 QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Manis pentadactyla isoform X3 ------------ ----- ---------MESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Manis javanica isoform X1 QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Manis javanica isoform X2 ------------ ----- ---------MESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Myotis brandtii QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Ochotona princeps QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Gorilla gorilla QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Pan troglodytes QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Rhinolophus ferrumequinum QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Saimiri boliviensis QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Ovis aries QAESLRYKLLGG LAVRR ACYGVLRFIMESGAKGCEVVVSGKL R GQRAKSMKFVDGLMIHS Table 4. 18SrRNA sequence alignment Human 18SrRNA 756-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-797 Camelus dromedarius 649-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-692 Mus musculus 656-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-698 Bos taurus 655-TGGA GG GCA AGT CTGGCGCCAGCAGCCG CG GTAA TT CCAGCT-697 Manis pentadactyla 659-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-701 Manis javanica 607-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-649 Ovis aries 655-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-697 Gorilla gorilla 655-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-697 Saimiri boliviensis 655-TGGA GG GCA AGT CTGGTGCCAGCAGCCG CG GTAA TT CCAGCT-697 Table 5. Ribosomal S30 sequence alignment Human S30 ribosomal protein 0-GRAKRRMQYNRRFVNVVPTFGKKKGPNANS-59 Camelus dromedarius 148-GQAKRRMQYNRRFVNVVPTFGK K KGPNANS-178 Camelus ferus 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Bos taurus 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Manis pentadactyla 133-GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Manis javanica 133-GRAKRRMQYNRRFVNVVPTFGKKKGPNANS-163 Rhinolophus ferrumequinum 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Ochotona princeps 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Myotis brandtii 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Mus musculus 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Pan paniscus 133- GRAKRRMQYNRRFVNVVPTFGK K KGPNANS-163 Cricetulus griseus 132- GRAKRRMQYNRRFVNVVPTFGK K KGPNAN–161 After figuring out all attachment sites which are available in table 1, we surveyed these points in normal cell state of translational system. 5VYC file from PDB site published by Lomakin and his colleagues in 2017 was used to achieve this goal ( 10 ). In this pdb file there is a 40S human ribosomal complex translating an mRNA. this complex gave us insights into internal bonds of ribosome, confirming that most of these points were involved with other ribosomal bases. For instance, in positions like G 625 , U 607 and A 605 from 18SrRNA, Lys 22 amio acid from S9 protein, Arg 143 from S3 protein and Asn 63 from S23 protein, NSP1 has made connections and interrupted previous bonds. (see table 6) Table 6. 18SrRNA interactions in normal cell state compared to Corona virus presence in cell Interaction points between NSp1 and 18SrRNA in the presence of coronavirus Interaction points of 18SrRNA in normal cell conditions G-600 C-622/U-630 G-601 G-6/C-621 A-604 C-603/C-639 A-605 C-603/C-638/G-598/Lys-22(Protein S9) G-606 NON U-607 Arg-143(Protein S3) C-624 G-623/G-617/U-631 G-625 Asn-63(Protein S23)/C-615/G-617 U-630 G-600 U-631 G-623/A-629/C-624 In Fig. 5 , three attachment points of Phe 124 , Glu 146 and Val 147 are shown being interrupted by NSP1 in S2 protein. These points form polar bonds instead with Ser 144 , Thr 149 , Ala 150 and Ile 151 in normal cell state, respectively. (see Fig. 5 ) In Fig. 6 , we showed S3 interactions with NSP1. Arg 116 from S3 has been interacting with several NSP1 amino acids while in cell normal state it had one polar bond with Cys 119 . (see Fig. 6 ) In case of other proteins which could attach to NSP1 in ribosomal complex, there was no recognizable interaction in 5VYC file. This could be the result of lack of crystallography information therefore other possible connections of NSP1 amino acids with 40S ribosomal complex members couldn’t be overruled Discussion The ribosomal system is fundamental to cell maintenance, growth, and replication by translating mRNA. SARSCoV-2 not only inhibits the translation of host cell mRNAs but also selectively promotes the translation of viral mRNAs, ultimately leading to host cell dysfunction and the release of new viral particles. Among the viral proteins, NSP1—an approximately 180–amino acid nonstructural protein—is primarily responsible for suppressing host mRNA translation. Crystallographic data have revealed that the carboxyl-terminal domain of NSP1 inserts into the mRNA entry channel of the 40S ribosomal subunit, thereby blocking host mRNAs from properly accessing the ribosome for translation( 20 ). In our study, we focused on dissecting the molecular interactions between NSP1 and the 40S ribosomal components. Our analyses identified several key interaction sites, notably in the 18S rRNA and ribosomal proteins S2, S3, and S30. Among these, the attachment points involved in the binding of the mRNA 5′end and the adjacent 18S rRNA regions appear to be crucial for regulating translation initiation. To gain further insight into these interactions, we compared the sequences of these molecules in humans with those from several potential intermediate host species—such as bats, pangolins, and camels—using Clustal Omega. The results demonstrated that sequences from these intermediate hosts contain distinguishable differences when compared to their human counterparts. For example, the S2 protein in camels and pangolins exhibits modifications that include point mutations and even deletion segments; these changes may disrupt the typical binding interface for NSP1. Phylogenetic analyses further indicated that the S2 protein from certain intermediate hosts groups differs, suggesting that these variations might underlie differences in viral interaction and pathogenicity( 11 , 12 , 13 , 15 ). In addition, previous studies by YuanQin Min and colleagues have highlighted that those essential residues—such as Arg-171 and Arg-175—in NSP1 are subject to modification in some coronavirus species, including MERSCoV( 18 ). In our analysis, residue 175 emerges as particularly significant because it not only contacts Lys-52 of the S30 protein but also interacts with adenine 605 and guanine 606 in the 18S rRNA. These interactions underscore the critical role of residue 175 in mediating NSP1’s stable attachment to the ribosomal complex. Although it is premature to definitively correlate these mutations with clinical outcomes, variations in this residue may influence the extent of translation suppression and thereby contribute to differences in disease severity between SARSCoV-2 and other coronaviruses. Overall, our findings provide clear evidence that NSP1 disrupts normal ribosomal interactions by competitively binding to key sites on the 18S rRNA and associated ribosomal proteins. This disruption of the translation machinery not only underlies the pathogenic potential of SARSCoV-2 but also highlights molecular differences that might explain why potential intermediate hosts remain asymptomatic carriers. These insights open new avenues for therapeutic interventions; for instance, targeting specific NSP1 mutations or designing small molecules to disrupt its binding to the ribosome might represent promising strategies for antiviral drug development or vaccine design. Declarations Clinical trial number not applicable Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Iran National Science Foundation (INSF) under project No. 4013516 and the National Institute of Genetic Engineering and Biotechnology (Grant No. 50000112 and 829). Author Contribution Dr.Bambai and Dr.Minuchehr conceived and designed the study. Afagh Bapirzadeh performed structural modeling of NSP1–40S ribosomal interactions and analyzed PDB data and carried out comparative sequence alignments and phylogenetic analyses across intermediate hosts. Afagh bapirzadeh contributed to bioinformatics pipeline development and result interpretation.Dr. Bambai and Dr.Minuchehr and Dr.Salehi supervised the project, secured funding, and provided critical revisions to study design. Afagh Bapirzadeh drafted the manuscript text; all authors prepared figures, reviewed the manuscript, and approved the final version for submission. Acknowledgement This work is based upon research funded by Iran National Science Foundation(INSF) under project NO.4013516 and also the National Institute of Genetic Engineering and Biotechnology (Grant No. 50000112 and 829). I am extremely grateful for their support, which enabled me to conduct this study and has contributed to significant advancements in the field Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References Abdi, M. Coronavirus disease 2019 (COVID-19) outbreak in Iran: Actions and problems. Infect. 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'SARS-CoV-2 nsp1: bioinformatics, potential structural and functional features, and implications for drug/vaccine designs'. Frontiers microbiology , 11. (2020). Semper, C., Watanabe, N. & Savchenko, A. Structural characterization of nonstructural protein 1 from SARS-CoV-2. iScience 24 (1), 101903. https://doi.org/10.1016/j.isci.2020.101903 (2021). Ganesh, B. et al. Epidemiology and pathobiology of SARS-CoV-2 (COVID-19) in comparison with SARS, MERS: An updated overview of current knowledge and future perspectives. Clin. Epidemiol. Global Health . 10 , 100694. https://doi.org/10.1016/j.cegh.2020.100694 (2021). Additional Declarations No competing interests reported. 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NSP1(red) and S3protein(blue) only polar contacts\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/6dd0c9bf3bd657bee15c724f.png"},{"id":92175500,"identity":"8b5e6d1c-f595-4c82-b187-a89c01005365","added_by":"auto","created_at":"2025-09-25 12:46:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266004,"visible":true,"origin":"","legend":"\u003cp\u003ea. NSP1(red) and S3(green)interactions (polar and non-polar contacts) right picture-3b. only polar contacts between NSP1(red) and S3(green) left picture\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/871b94669cba4e98ffc4dd53.png"},{"id":92175498,"identity":"f67503c6-1706-4657-bd9b-afd5d754432b","added_by":"auto","created_at":"2025-09-25 12:46:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":205550,"visible":true,"origin":"","legend":"\u003cp\u003ea. NSP1(red) and S2 (purple)interactions, right picture.4b. NSP1(red) and S2(purple) only polar contacts, left picture.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/778f30f53124a3653a021969.png"},{"id":92176876,"identity":"ef8d0fa0-8aba-4b3d-a94a-283dd31bfd58","added_by":"auto","created_at":"2025-09-25 12:54:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114662,"visible":true,"origin":"","legend":"\u003cp\u003einternal interactions of S2 in normal cell state (both colors show different parts of the same protein)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/a2b58e089776cf88a2c471bf.png"},{"id":92176878,"identity":"56049cc1-2149-4a8a-86c3-21adbcbb9413","added_by":"auto","created_at":"2025-09-25 12:54:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24733,"visible":true,"origin":"","legend":"\u003cp\u003einternal interactions of S3 in normal cell state (both colors show different parts of the same protein)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/aed2939c8717d688386d6d08.png"},{"id":93674994,"identity":"18c9df05-14a7-4254-8b72-02470ba0ce0f","added_by":"auto","created_at":"2025-10-16 10:53:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2140718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/74193f2e-5133-409b-adea-4390c52e79bf.pdf"},{"id":92176883,"identity":"b19d00e8-4a66-4755-ae1d-346df62b5ae0","added_by":"auto","created_at":"2025-09-25 12:54:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":154725,"visible":true,"origin":"","legend":"","description":"","filename":"ribosomalS30S3andS2alignmentsincarrieranimals.docx","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/e566c3e746756981cf62d2b2.docx"},{"id":92175493,"identity":"9089b070-4b46-4d7e-982d-d2d85ddb9ae5","added_by":"auto","created_at":"2025-09-25 12:46:55","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":265646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7546879/v1/da0d4b9cf257a6c7c1d620ed.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption","fulltext":[{"header":"Key Points","content":"\u003cp\u003eNSP1 interactions in human 40S ribosomal complex and involved proteins\u003c/p\u003e\n\u003cp\u003eComparison between human ribosomal sequences and protein sequences and intermediate animals\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAccording to the World Health Organization (WHO) statement in March 2020, the rapid global spread of coronavirus disease (COVID-19) led to its declaration as a global pandemic (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In response, the Health Ministry of the Islamic Republic of Iran, together with the government, announced a national quarantine and established a specialized committee tasked with implementing social education and prevention strategies. Among the various measures, authorities encouraged researchers and provided funding for projects focused on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19. Notably, the Iran National Institute of Genetic Engineering and Biotechnology has mobilized several research groups to investigate different aspects of this disease(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCOVID-19 is initiated when the SARS-CoV-2 virus enters human host cells via the binding of its structural spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The viral genome is composed of a single positive-sense RNA that encodes 11 open reading frames (ORFs). Following transcription and translation, these ORFs yield several polyproteins that are essential for viral replication and survival. These polyproteins are subsequently processed by viral proteases, including the main protease (Mpro), the papain-like protease (PLP), and the 3C-like protease (3CLpro). One of the resulting proteins, non-structural protein 1 (NSP1), is a 20 kDa factor localized in the cytoplasm of infected cells(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Yoshimoto has suggested that NSP1, also referred to as the leader protein, plays a crucial role in inhibiting host cell gene expression, likely by binding to the 40S ribosomal subunits(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This protein, which is also called the host shutoff factor, suppresses host innate immune functions(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRibosomes are essential cellular organelles composed of protein and RNA subunits that are responsible for protein synthesis. For NSP1 to exert its function, it must bind to the 40S ribosomal subunit\u003c/p\u003e\u003cp\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This binding is thought to influence the ribosome\u0026rsquo;s architecture by disrupting the intrinsic interactions among its proteins and RNA(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). We hypothesize that when NSP1 engages with ribosomal subunit components, it perturbs both internal and external contacts\u0026mdash;potentially causing contact inhibition between adjacent nucleotides or proximate amino acids\u0026mdash;and thereby impairs normal ribosomal function. According to Schubert and colleagues, the full-length 5\u0026prime; untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe novel coronavirus is transmitted to humans via potential intermediate hosts. Reservoir species, such as horseshoe bats, have experienced multiple recombination events among different SARS-related coronavirus ancestors (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Other animal species, including \u003cem\u003epangolins\u003c/em\u003e and \u003cem\u003emasked palm civets\u003c/em\u003e, have also been proposed as potential intermediaries in the transmission chain(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In addition, studies conducted in New Zealand on \u003cem\u003erabbits\u003c/em\u003e, \u003cem\u003eferrets\u003c/em\u003e, and \u003cem\u003ehamsters\u003c/em\u003e have shown that although these animals can harbor the virus asymptomatically, they do not serve as true reservoirs(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). It is noteworthy that these potential intermediate hosts, even when in contact with the virus, are neither diagnosed as infected nor do they exhibit clinical symptoms(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In this article, we demonstrate that NSP1 interactions with the human 40S ribosomal complex differ markedly from those observed in potential intermediate hosts. In these animals, modifications such as deletions, insertions, or other sequence variations in ribosomal proteins result in altered binding interfaces with the viral NSP1. Such changes likely lead to weaker or even absent interactions, which may contribute to their status as carriers without manifesting disease.\u003c/p\u003e\u003cp\u003eTo examine this hypothesis, we first analyzed the structure of NSP1 and detailed its interactions with the 40S human ribosome, identifying critical hotspot sites. Subsequently, we compared these interaction regions between humans and potential intermediate hosts\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003eIn December 2020, Shi and colleagues determined the crystallographic structure of the SARS-CoV-2 NSP1 protein in complex with the 40S ribosome(\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e). This structure, available from the Protein Data Bank under the identifier 7K5i, forms the fundamental basis of our study(. We utilized the free educational version of PyMOL (version 2.4.1) to examine the binding interface between NSP1 and the human 40S ribosomal complex. Specifically, we investigated interactions between NSP1 and four key components: the 18S rRNA and the ribosomal proteins S30, S3, and S2 (see Table\u0026nbsp;1). For detection of contacts, we applied a distance cutoff of 4 \u0026Aring; to define both polar (e.g., hydrogen bonds) and non-polar interactions using PyMOL\u0026rsquo;s measurement tools and built-in interaction analysis features.\u003c/p\u003e\n\u003cp\u003eTable 1. NSP1 interactions with 40S human ribosome (18SrRNA and S2, S3, S30). polar contacts are shown with star\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eGiven the multiple interactions between NSP1 and the human ribosome, we extended our analysis to compare the relevant ribosomal sequences between humans and several potential intermediate host species(17,18,19). The species examined include bats (genus \u003cem\u003eRhinolophus\u003c/em\u003e and \u003cem\u003eMyotis\u003c/em\u003e), pangolins (\u003cem\u003eManis pentadactyla\u003c/em\u003e and \u003cem\u003eManis javanica\u003c/em\u003e), the American pika (\u003cem\u003eOchotona princeps\u003c/em\u003e), and camels (\u003cem\u003eCamelus dromedarius\u003c/em\u003e and \u003cem\u003eCamelus ferus\u003c/em\u003e). In addition, species with close evolutionary relationships to humans\u0026mdash;such as the mouse (\u003cem\u003eMus musculus\u003c/em\u003e), cow (\u003cem\u003eBos taurus\u003c/em\u003e), monkeys (genera \u003cem\u003eMandrillus\u003c/em\u003e and \u003cem\u003eSaimiri\u003c/em\u003e), gorilla (\u003cem\u003eGorilla gorilla\u003c/em\u003e), chimpanzee (\u003cem\u003ePan troglodytes\u003c/em\u003e), and sheep (\u003cem\u003eOvis aries\u003c/em\u003e)were included in the sequence alignments(20).\u003c/p\u003e\n\u003cp\u003eTo perform these alignments, we initially retrieved the NSP1-interacting protein and ribosomal RNA sequences from the National Center for Biotechnology Information (NCBI) database. Multiple sequence alignments were then executed using Clustal Omega, a freely available, non-commercial web tool. The results of these alignments, which served to highlight differences in the putative NSP1 interaction sites between humans and the examined species, are summarized in Tables\u0026nbsp;2, 3, 4, and 5. It is important to note that in some cases data availability was limited due to incomplete protein or genome sequences in public databases.This integrated methodological approach enabled us to systematically compare the interaction interfaces of NSP1 with 40S ribosomal components across species. The insights garnered from these analyses provide a potential explanation for the differential susceptibility to SARS-CoV-2 infection observed between humans and various intermediate hosts.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn table 1 the most polar interactions are between NSP1 protein and 18SRNA. 18SRNA is the biggest subunit of 40S ribosome. The interactions between 18SRNA and NSP1 are not only polar but also consist of four non-polar bonds under 4 angstroms (A\u0026deg;). NSP1 at Lys-164, His-165, Ser-167, Arg-171, Arg-175 amino acids have polar contact with 18SRNA nucleotide bases. Among this Arg-175 from NSP1 has polar bonds with A\u003csup\u003e605\u003c/sup\u003eand G\u003csup\u003e606\u003c/sup\u003e, Arg-171 with G\u003csup\u003e600,601\u003c/sup\u003e and U\u003csup\u003e607\u003c/sup\u003e. Lys-164, His-165 and Ser-167 have both polar and non-polar bonds with 18SRNA nucleotides. Ser-167 has a polar contact with G\u003csup\u003e600\u003c/sup\u003e but a non-polar contact with G\u003csup\u003e601\u003c/sup\u003e. His-165 also has polar bond with U\u003csup\u003e630\u003c/sup\u003e and a non-polar bond with U\u003csup\u003e607\u003c/sup\u003e. In Lys-164 case as it can be seen in Table\u0026nbsp;1, there are two polar bonds with G\u003csup\u003e625\u003c/sup\u003eand C\u003csup\u003e624\u003c/sup\u003eand one non-polar bond with U\u003csup\u003e631\u003c/sup\u003e. Also Glu-172 has one non-polar bond with U\u003csup\u003e607\u003c/sup\u003e base of 18SRNA. (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eAmong proteins involved in ribosomal translation system helping 40S subunits and interacting NSP1, there were three specific protein S2, S3 and S30. S30 has the lowest connection to NSP1, one polar contact between Lys\u003csup\u003e52\u003c/sup\u003e from S30 protein with Glu-176 and a non-polar contact between Lys\u003csup\u003e52\u003c/sup\u003e and Arg-175 from NSP1 protein (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe last protein which will be discussed is S2 from 40S ribosomal complex. NSP1 in Asn-178 and Gln-158 form polar contacts with Val\u003csup\u003e106\u003c/sup\u003e and Val\u003csup\u003e147\u003c/sup\u003e, respectively. These two polar bonds were the only polar contacts between NSP1 and S2, other contacts were all non-polar. Gln-158 in three positions of Glu\u003csup\u003e146\u003c/sup\u003e, Val\u003csup\u003e147\u003c/sup\u003e and Phe\u003csup\u003e124\u003c/sup\u003ehad non-polar contacts. Trp-161 from NSP1 had non-polar connections to Phe\u003csup\u003e124\u003c/sup\u003e, Thr\u003csup\u003e122\u003c/sup\u003e and Pro\u003csup\u003e111\u003c/sup\u003e. Phe-157 also had non-polar connections with Phe\u003csup\u003e124\u003c/sup\u003eand Ile\u003csup\u003e109\u003c/sup\u003efrom S2 protein. In addition to Asn-178, Met-174 also had one non-polar bond with Val\u003csup\u003e106\u003c/sup\u003efrom NSP1. (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eThe next step in finding NSP1 interactions with ribosomal complex was alignment among human 18SrRNA and its dependent proteins with intermediate animals. One of the most significant differences in attachment sites of NSP1 with human ribosomes compared to camel, bats and pangolins specifically were seen in S2 protein. Camel S2 protein sequence (\u003cem\u003eCamelus dromedaries\u003c/em\u003e) which is shown in Table\u0026nbsp;2, had a relatively huge deletion, starting from amino acid number 48 and lasts to amino acid number 80. In between this deleted segment, there were two important amino acids, Glu-146 and Val-147 which were NSP1 attachment points to human S2 ribosome. This deletion doesn\u0026rsquo;t exist in similar sequences of human ribosomal protein S2.Another two differences come from camel S2 protein amino acids 34 and 37 in which instead of Arg and Phe, Glu and Leu were replaced. It is important to mention that camel is one of Corona virus intermediate animals who don\u0026rsquo;t get infected by the virus and is only a carrier. S2 protein of isoform X1. a type of monkey (\u003cem\u003eMandrillus leucophaeus\u003c/em\u003e) also had Threonine instead of Isoleucine in the position of 109. (see Table\u0026nbsp;2)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. ribosomal S2 sequence alignment\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHuman S2 ribosomal protein\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cspan class=\"Underline\"\u003eV\u003c/span\u003eLK\u003cspan class=\"Underline\"\u003eI\u003c/span\u003eM\u003cspan class=\"Underline\"\u003eP\u003c/span\u003eVQKQTRAGQR\u003cspan class=\"Underline\"\u003eT\u003c/span\u003eR\u003cspan class=\"Underline\"\u003eF\u003c/span\u003eKAFVAIGDYNGHVGLGVKCSK\u003cspan class=\"Underline\"\u003eEV\u003c/span\u003eATAIR\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus dromedarius\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQ\u003cstrong\u003eQT\u003c/strong\u003eR\u003cspan class=\"BoldUnderline\"\u003eL\u003c/span\u003eKAFVATGDDKG----------\u003cstrong\u003e--\u003c/strong\u003e-----\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus bactrianus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMus musculus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBos taurus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis javanica\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDD\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGHYNGHVGLGX\u0026mdash;SK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMyotis brandtii\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOchotona princeps\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGorilla gorilla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMandrillus leucophaeus isoform X1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eT\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMandrillus leucophaeus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePan paniscus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRhinolophus ferrumequinm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSaimiri boliviensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKEFVAIGDYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOvis aries\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLKDE\u003cstrong\u003eV\u003c/strong\u003eLK\u003cstrong\u003eI\u003c/strong\u003eM\u003cstrong\u003eP\u003c/strong\u003eVQKQTRAGQR\u003cstrong\u003eT\u003c/strong\u003eR\u003cstrong\u003eF\u003c/strong\u003eKAFVAIADYNGHVGLGVKCSK\u003cstrong\u003eEV\u003c/strong\u003eATAIR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eS3 ribosomal protein similar to S2 protein showed differences between human and intermediate animals. For example, a 126 amino acids deleted segment at the beginning of this protein in X2 and X3 isoforms of \u003cem\u003eManis pentadactyla\u003c/em\u003e and \u003cem\u003eManis javanica\u003c/em\u003e existed. This deletion was responsible for protein size differences. Aside from this fact, five attachment points in NSP1 protein including Leu-113, Ala-114, Val-115, Arg-116 and Arg-117 were also deleted (see table 3).\u003c/p\u003e\n\u003cp\u003eSequence Alignments between 18SrRNA of human 40S ribosomal and intermediate animals showed no significant differences and all attachment points to NSP1 existed for all of them (see table 4). This also applied for S30 protein. Having only 59 amino acids length in human S30 protein in contrast with longer length of this protein in intermediate animals was a very important fact (see table 5).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\n\u003cp\u003eTable 3. ribosomal S3 sequence alignment\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHuman S3 ribosomal protein\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cspan class=\"Underline\"\u003eLAVRR\u003c/span\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cspan class=\"Underline\"\u003eR\u003c/span\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus dromedarius\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMus musculus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBos taurus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla isoform X1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla isoform X2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla isoform X3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e------------\u003cstrong\u003e-----\u003c/strong\u003e---------MESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis javanica isoform X1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis javanica isoform X2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e------------\u003cstrong\u003e-----\u003c/strong\u003e---------MESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMyotis brandtii\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOchotona princeps\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGorilla gorilla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePan troglodytes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRhinolophus ferrumequinum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSaimiri boliviensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOvis aries\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQAESLRYKLLGG\u003cstrong\u003eLAVRR\u003c/strong\u003eACYGVLRFIMESGAKGCEVVVSGKL\u003cstrong\u003eR\u003c/strong\u003eGQRAKSMKFVDGLMIHS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\n\u003cp\u003eTable 4. 18SrRNA sequence alignment\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabc\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHuman 18SrRNA\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e756-TGGA\u003cspan class=\"Underline\"\u003eGG\u003c/span\u003eGCA\u003cspan class=\"Underline\"\u003eAGT\u003c/span\u003eCTGGTGCCAGCAGCCG\u003cspan class=\"Underline\"\u003eCG\u003c/span\u003eGTAA\u003cspan class=\"Underline\"\u003eTT\u003c/span\u003eCCAGCT-797\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus dromedarius\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e649-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-692\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMus musculus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e656-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-698\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBos taurus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e655-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGCGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-697\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e659-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-701\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis javanica\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e607-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-649\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOvis aries\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e655-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-697\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGorilla gorilla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e655-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-697\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSaimiri boliviensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e655-TGGA\u003cstrong\u003eGG\u003c/strong\u003eGCA\u003cstrong\u003eAGT\u003c/strong\u003eCTGGTGCCAGCAGCCG\u003cstrong\u003eCG\u003c/strong\u003eGTAA\u003cstrong\u003eTT\u003c/strong\u003eCCAGCT-697\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\n\u003cp\u003eTable 5. Ribosomal S30 sequence alignment\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabd\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHuman S30 ribosomal protein\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0-GRAKRRMQYNRRFVNVVPTFGKKKGPNANS-59\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus dromedarius\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e148-GQAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-178\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCamelus ferus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBos taurus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis pentadactyla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133-GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eManis javanica\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133-GRAKRRMQYNRRFVNVVPTFGKKKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRhinolophus ferrumequinum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOchotona princeps\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMyotis brandtii\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMus musculus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePan paniscus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e133- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNANS-163\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCricetulus griseus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e132- GRAKRRMQYNRRFVNVVPTFGK\u003cstrong\u003eK\u003c/strong\u003eKGPNAN\u0026ndash;161\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAfter figuring out all attachment sites which are available in table 1, we surveyed these points in normal cell state of translational system. 5VYC file from PDB site published by Lomakin and his colleagues in 2017 was used to achieve this goal (\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e). In this pdb file there is a 40S human ribosomal complex translating an mRNA. this complex gave us insights into internal bonds of ribosome, confirming that most of these points were involved with other ribosomal bases. For instance, in positions like G\u003csup\u003e625\u003c/sup\u003e, U\u003csup\u003e607\u003c/sup\u003eand A\u003csup\u003e605\u003c/sup\u003e from 18SrRNA, Lys\u003csup\u003e22\u003c/sup\u003eamio acid from S9 protein, Arg\u003csup\u003e143\u003c/sup\u003efrom S3 protein and Asn\u003csup\u003e63\u003c/sup\u003e from S23 protein, NSP1 has made connections and interrupted previous bonds. (see table 6)\u003c/p\u003e\n\u003cp\u003eTable 6. 18SrRNA interactions in normal cell state compared to Corona virus presence in cell\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tabe\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eInteraction points between NSp1 and 18SrRNA in the presence of coronavirus\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eInteraction points of 18SrRNA in normal cell conditions\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eG-600\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC-622/U-630\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eG-601\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG-6/C-621\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eA-604\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC-603/C-639\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eA-605\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC-603/C-638/G-598/Lys-22(Protein S9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eG-606\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNON\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eU-607\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArg-143(Protein S3)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eC-624\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG-623/G-617/U-631\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eG-625\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAsn-63(Protein S23)/C-615/G-617\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eU-630\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG-600\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eU-631\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eG-623/A-629/C-624\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, three attachment points of Phe\u003csup\u003e124\u003c/sup\u003e, Glu\u003csup\u003e146\u003c/sup\u003eand Val\u003csup\u003e147\u003c/sup\u003eare shown being interrupted by NSP1 in S2 protein. These points form polar bonds instead with Ser\u003csup\u003e144\u003c/sup\u003e, Thr\u003csup\u003e149\u003c/sup\u003e, Ala\u003csup\u003e150\u003c/sup\u003eand Ile\u003csup\u003e151\u003c/sup\u003e in normal cell state, respectively. (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, we showed S3 interactions with NSP1. Arg\u003csup\u003e116\u003c/sup\u003e from S3 has been interacting with several NSP1 amino acids while in cell normal state it had one polar bond with Cys\u003csup\u003e119\u003c/sup\u003e. (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In case of other proteins which could attach to NSP1 in ribosomal complex, there was no recognizable interaction in 5VYC file. This could be the result of lack of crystallography information therefore other possible connections of NSP1 amino acids with 40S ribosomal complex members couldn\u0026rsquo;t be overruled\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ribosomal system is fundamental to cell maintenance, growth, and replication by translating mRNA. SARSCoV-2 not only inhibits the translation of host cell mRNAs but also selectively promotes the translation of viral mRNAs, ultimately leading to host cell dysfunction and the release of new viral particles. Among the viral proteins, NSP1\u0026mdash;an approximately 180\u0026ndash;amino acid nonstructural protein\u0026mdash;is primarily responsible for suppressing host mRNA translation. Crystallographic data have revealed that the carboxyl-terminal domain of NSP1 inserts into the mRNA entry channel of the 40S ribosomal subunit, thereby blocking host mRNAs from properly accessing the ribosome for translation(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our study, we focused on dissecting the molecular interactions between NSP1 and the 40S ribosomal components. Our analyses identified several key interaction sites, notably in the 18S rRNA and ribosomal proteins S2, S3, and S30. Among these, the attachment points involved in the binding of the mRNA 5\u0026prime;end and the adjacent 18S rRNA regions appear to be crucial for regulating translation initiation. To gain further insight into these interactions, we compared the sequences of these molecules in humans with those from several potential intermediate host species\u0026mdash;such as bats, pangolins, and camels\u0026mdash;using Clustal Omega. The results demonstrated that sequences from these intermediate hosts contain distinguishable differences when compared to their human counterparts. For example, the S2 protein in camels and pangolins exhibits modifications that include point mutations and even deletion segments; these changes may disrupt the typical binding interface for NSP1. Phylogenetic analyses further indicated that the S2 protein from certain intermediate hosts groups differs, suggesting that these variations might underlie differences in viral interaction and pathogenicity(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, previous studies by YuanQin Min and colleagues have highlighted that those essential residues\u0026mdash;such as Arg-171 and Arg-175\u0026mdash;in NSP1 are subject to modification in some coronavirus species, including MERSCoV(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In our analysis, residue 175 emerges as particularly significant because it not only contacts Lys-52 of the S30 protein but also interacts with adenine 605 and guanine 606 in the 18S rRNA. These interactions underscore the critical role of residue 175 in mediating NSP1\u0026rsquo;s stable attachment to the ribosomal complex. Although it is premature to definitively correlate these mutations with clinical outcomes, variations in this residue may influence the extent of translation suppression and thereby contribute to differences in disease severity between SARSCoV-2 and other coronaviruses.\u003c/p\u003e\u003cp\u003eOverall, our findings provide clear evidence that NSP1 disrupts normal ribosomal interactions by competitively binding to key sites on the 18S rRNA and associated ribosomal proteins. This disruption of the translation machinery not only underlies the pathogenic potential of SARSCoV-2 but also highlights molecular differences that might explain why potential intermediate hosts remain asymptomatic carriers. These insights open new avenues for therapeutic interventions; for instance, targeting specific NSP1 mutations or designing small molecules to disrupt its binding to the ribosome might represent promising strategies for antiviral drug development or vaccine design.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003cp\u003enot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Iran National Science Foundation (INSF) under project No. 4013516 and the National Institute of Genetic Engineering and Biotechnology (Grant No. 50000112 and 829).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDr.Bambai and Dr.Minuchehr conceived and designed the study. Afagh Bapirzadeh performed structural modeling of NSP1\u0026ndash;40S ribosomal interactions and analyzed PDB data and carried out comparative sequence alignments and phylogenetic analyses across intermediate hosts. Afagh bapirzadeh contributed to bioinformatics pipeline development and result interpretation.Dr. Bambai and Dr.Minuchehr and Dr.Salehi supervised the project, secured funding, and provided critical revisions to study design. Afagh Bapirzadeh drafted the manuscript text; all authors prepared figures, reviewed the manuscript, and approved the final version for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work is based upon research funded by Iran National Science Foundation(INSF) under project NO.4013516 and also the National Institute of Genetic Engineering and Biotechnology (Grant No. 50000112 and 829). I am extremely grateful for their support, which enabled me to conduct this study and has contributed to significant advancements in the field\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdi, M. Coronavirus disease 2019 (COVID-19) outbreak in Iran: Actions and problems. \u003cem\u003eInfect. Control Hosp. Epidemiol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 754\u0026ndash;755 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArtika, I., Made, A. K. \u0026amp; Dewantari and Ageng Wiyatno. 'Molecular biology of coronaviruses: current knowledge', \u003cem\u003eHeliyon\u003c/em\u003e: e04743. (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConnor, R. F. \u0026amp; Rachel, L. R. 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Global Health\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 100694. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cegh.2020.100694\u003c/span\u003e\u003cspan address=\"10.1016/j.cegh.2020.100694\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, NSP1, Bioinformatics, 40s ribosomal subunits, Bats, Manis","lastPublishedDoi":"10.21203/rs.3.rs-7546879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7546879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNSP1 protein is one of the first produced proteins of SARS-CoV-2, the virus which is responsible for Corona disease, sited at the beginning of 5\u003csup\u003é\u003c/sup\u003e end of virus gene number one. NSP1 bonds to Human 40S ribosomal subunits has potential roles in host cells mRNA translation inhibition.to clarify subtle molecular reasons of carrier animal\u0026rsquo;s pathogenicity, we used bioinformatics tools and 7k5i file structure acquired from PDB data bank to investigate interactions and internal bonds of 40S ribosomes in normal cell conditions. NSP1 protein bonds in complex with ribosomes of Human host cells in individuals affected by SARS-CoV-2 differ from normal states. Majority of NSP1 interactions involve 18SrRNA ribosomal subunit and one of ribosomal proteins. These bonds are at A\u003csup\u003e605\u003c/sup\u003eand G\u003csup\u003e600,601\u003c/sup\u003e nucleotide positions in 18SrRNA leading to both physically and spatially disrupting existing internal interactions in this subunit which eventually could cause ribosome to lose its function. Additional to 18SrRNA, NSP1 also bonds with three ribosomal proteins: S2, S3 and S30.alignment comparison of these molecules in Human and other mammalian in one hand and between Human and carrier animals such as camel and Manis in another hand showed for example camel S2 protein is more like Manis instead of being genetically close to human or cows. These crucial findings strongly support important role of NSP1 in translation inhibition of host cells ribosomes.by using these results and making a few structural changes at carboxyl end of NSP1 protein suppression and restriction of cancerous cell growth can be gained.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption Comparative Analysis of NSP1–40S Ribosomal Interactions Across Intermediate Hosts: Implications for Host Translation Disruption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 12:46:50","doi":"10.21203/rs.3.rs-7546879/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b873cab2-1e7b-48eb-9bd0-3dac1050a238","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55110784,"name":"Biological sciences/Biochemistry"},{"id":55110785,"name":"Biological sciences/Cell biology"},{"id":55110786,"name":"Biological sciences/Microbiology"},{"id":55110787,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-10-16T10:53:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 12:46:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7546879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7546879","identity":"rs-7546879","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}