Complete Alanine Scanning of Intersubunit Interfaces in a Foot-and-Mouth Disease Virus Capsid Reveals Critical Contributions of Many Side Chains to Particle Stability and Viral Function
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m304990200
ISSN1083-351X
AutoresRoberto Mateo, A. Díaz Díaz, Éric Baranowski, Mauricio G. Mateu,
Tópico(s)Bacteriophages and microbial interactions
ResumoSpherical virus capsids are large, multimeric protein shells whose assembly and stability depend on the establishment of multiple non-covalent interactions between many polypeptide subunits. In a foot-and-mouth disease virus capsid, 42 amino acid side chains per protomer are involved in noncovalent interactions between pentameric subunits that function as assembly/disassembly intermediates. We have individually truncated to alanine these 42 side chains and assessed their relevance for completion of the virus life cycle and capsid stability. Most mutations provoked a drastic reduction in virus yields. Nearly all of these critical mutations led to virions whose thermal inactivation rates differed from that of the parent virus, and many affected also early steps in the viral cycle. Rapid selection of genotypic revertants or variants with forward or compensatory mutations that restored viability was occasionally detected. The results with this model virus indicate the following. (i) Most of the residues at the interfaces between capsid subunits are critically important for viral function, in part but not exclusively because of their involvement in intersubunit recognition. Each hydrogen bond and salt bridge buried at the subunit interfaces may be important for capsid stability. (ii) New mutations able to restore viability may arise frequently at the subunit interfaces during virus evolution. (iii) A few interfacial side chains are functionally tolerant to truncation and may provide adequate mutation sites for the engineering of a thermostable capsid, potentially useful as an improved vaccine. Spherical virus capsids are large, multimeric protein shells whose assembly and stability depend on the establishment of multiple non-covalent interactions between many polypeptide subunits. In a foot-and-mouth disease virus capsid, 42 amino acid side chains per protomer are involved in noncovalent interactions between pentameric subunits that function as assembly/disassembly intermediates. We have individually truncated to alanine these 42 side chains and assessed their relevance for completion of the virus life cycle and capsid stability. Most mutations provoked a drastic reduction in virus yields. Nearly all of these critical mutations led to virions whose thermal inactivation rates differed from that of the parent virus, and many affected also early steps in the viral cycle. Rapid selection of genotypic revertants or variants with forward or compensatory mutations that restored viability was occasionally detected. The results with this model virus indicate the following. (i) Most of the residues at the interfaces between capsid subunits are critically important for viral function, in part but not exclusively because of their involvement in intersubunit recognition. Each hydrogen bond and salt bridge buried at the subunit interfaces may be important for capsid stability. (ii) New mutations able to restore viability may arise frequently at the subunit interfaces during virus evolution. (iii) A few interfacial side chains are functionally tolerant to truncation and may provide adequate mutation sites for the engineering of a thermostable capsid, potentially useful as an improved vaccine. Assembly and stability of the multimeric proteins that constitute icosahedral virus capsids depend on the occurrence of multiple non-covalent interactions between many polypeptide subunits (1Liljas L. Prog. Biophys. Mol. Biol. 1986; 48: 1-36Crossref PubMed Scopus (43) Google Scholar, 2Rossmann M.G. Johnson J.E. Annu. Rev. Biochem. 1989; 58: 533-573Crossref PubMed Google Scholar). Compared with small oligomeric proteins, the adaptive structural solutions for subunit recognition and stability may be uniquely limited in virus capsids. 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However, to our knowledge no systematic mutagenesis approach to experimentally dissect the contribution of each residue in the subunit interfaces to the stability or functionality of a virus capsid or any other large protein assembly had been attempted to date. Foot-and-mouth disease virus (FMDV), 1The abbreviations used are: FMDV, foot-and-mouth disease virus; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BHK, baby hamster kidney; FMD, foot-and-mouth disease.1The abbreviations used are: FMDV, foot-and-mouth disease virus; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BHK, baby hamster kidney; FMD, foot-and-mouth disease. a picornavirus for which much structural and functional information is available, provides a simple model for the dissection of the molecular determinants of assembly, stability, and disassembly of viral capsids and other large protein complexes. Moreover, FMDV causes the economically most important disease of farm animals (32Sobrino F. Saiz M. Jiménez-Clavero M.A. Núñez J.I. Rosas M.F. Baranowski E. Ley V. Vet. Res. 2001; 32: 1-30Crossref PubMed Scopus (224) Google Scholar). The recent epizootics in the United Kingdom, with estimated losses that could exceed £10 billion in that country alone (33Samuel A.R. Knowles N.J. Trends Genet. 2001; 17: 421-424Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), have provided a stark remainder within the European Union of the devastating economic and social damage FMDV may cause worldwide. One important problem of the current FMD vaccines, based on chemically inactivated virions, is their very low stability. Vaccines based on particles with improved thermostability would be highly desirable in the prevention of FMD and other viral diseases. Assembly of the picornaviral capsid proceeds in several steps (34Rueckert R.R. Fields B. Knipe D.M. Howley P.M. Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 609-654Google Scholar). The capsid proteins VP0 (1AB), VP3 (1C), and VP1 (1D) are translated as a polyprotein precursor (P1), may fold co-translationally (2Rossmann M.G. Johnson J.E. Annu. Rev. Biochem. 1989; 58: 533-573Crossref PubMed Google Scholar), and are proteolytically processed to yield the mature protomer. Five protomers are assembled to form a pentameric intermediate, and finally, 12 pentamers are assembled to form the icosahedral capsid (Fig. 1). After encapsidation of the RNA genome most VP0 molecules are processed to give VP4 (1A, the N terminus of VP0) and VP2 (1B). Disassembly of the FMDV virion in vivo begins with its dissociation into pentamers (35Vasquez C. Denoya C.D. La Torre J.L. Palma E. Virology. 1979; 97: 195-200Crossref PubMed Scopus (29) Google Scholar) by acidification in the endosomes (36Carrillo E.C. Giachetti C. Campos R.H. Virology. 1984; 135: 542-545Crossref PubMed Scopus (41) Google Scholar). Mild heating of FMDV virions also leads to irreversible dissociation into stable pentamers (34Rueckert R.R. Fields B. Knipe D.M. Howley P.M. Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 609-654Google Scholar), an event that appears as the main cause for the need of a cold chain to preserve FMD vaccines. Analysis of the crystal structure of the FMDV capsid (37Acharya R. Fry E. Stuart D. Fox G. Rowlands D. Brown F. Nature. 1989; 337: 709-716Crossref PubMed Scopus (673) Google Scholar, 38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 39Lea S. Abu-Ghazaleh R. Blakemore W. Curry S. Fry E. Jackson T. King A. Logan D. Newman J. Stuart D. Structure. 1995; 3: 571-580Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 40Curry S. Fry E. Blakemore W. Abu-Ghazaleh R. Jackson T. King A. Lea S. Newman J. Rowlands D. Stuart D. Structure. 1996; 4: 135-145Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 41Fry E.E. Lea S.M. Jackson T. Newman J.W. Ellard F.M. Blakemore W.E. Abu-Ghazaleh R. Samuel A. King A.M. Stuart D.I. EMBO J. 1999; 18: 543-554Crossref PubMed Scopus (278) Google Scholar) indicates that the pentameric intermediate subunits interact mainly through a relatively limited number of electrostatic interactions; a role of His-142 of VP3 in the acid-induced disassembly of FMDV has already been demonstrated (20Ellard F.M. Drew J. Blakemore W.E. Stuart D.I. King A.M. J. Gen. Virol. 1999; 80: 1911-1918Crossref PubMed Scopus (96) Google Scholar). In the present study, the functional importance of each amino acid side chain involved in noncovalent interactions between subunits in a virus protein shell has been tested by alanine-scanning mutagenesis of the FMDV capsid. The results reveal severe intolerance to variation due to the involvement of many interfacial residues in capsid stability and in other steps of the virus life cycle, and identify those tolerant residues that could be replaced for the rational engineering of a thermostable capsid. Viruses and Plasmids—FMDV C-S8c1 is a plaque-purified derivative of serotype C isolate C1SantaPau-Sp70 (42Sobrino F. Dávila M. Ortín J. Domingo E. Virology. 1983; 128: 310-318Crossref PubMed Scopus (224) Google Scholar). Plasmids pO1K/C-S8c1, pO1K/Δ3242, and p3242/C-S8c1 have been described (43Baranowski E. Sevilla N. Verdaguer N. Ruiz-Jarabo C. Beck E. Domingo E. J. Virol. 1998; 72: 6362-6372Crossref PubMed Google Scholar). Use of a cDNA clone entirely derived from a type C virus had been hampered by its low infectivity. Instead, pO1K/C-S8c1 was derived from an infectious cDNA clone obtained by Beck and co-workers (44Zibert A. Maass G. Strebel K. Falk M.M. Beck E. J. Virol. 1990; 64: 2467-2473Crossref PubMed Google Scholar) from FMDV O1 Kaufbeuren. pO1K/C-S8c1 contains a cDNA copy of an infectious chimeric FMDV genome coding for the capsid proteins and protease 2A of C-S8c1, a chimeric 2B protein and all of the nonstructural proteins (2A and 2B excepted) of O1Kaufbeuren. Plasmid pO1K/Δ3242 corresponds to pO1K/C-S8c1 with a segment of 3242 bp (between the two NgoMIV restriction sites) deleted. This segment includes the entire region coding for the capsid proteins of FMDV C-S8c1. Plasmid p3242/C-S8c1 contains this same 3242-bp segment inserted in a vector derived from plasmid pGEM-5Zf(+) (43Baranowski E. Sevilla N. Verdaguer N. Ruiz-Jarabo C. Beck E. Domingo E. J. Virol. 1998; 72: 6362-6372Crossref PubMed Google Scholar). Site-directed Mutagenesis, Subcloning, and DNA Sequencing—Site-directed mutagenesis of FMDV capsid residues was carried out on plasmid p3242/C-S8c1 by the inverse PCR method using the QuikChange system (Stratagene) and pairs of 25- to 36-mer oligonucleotides. The mutations were checked by dideoxynucleotide-based automated sequencing. No second site mutations were found. The mutagenized 3242-bp NgoMIV segments were subcloned in pO1K/Δ3242 to obtain pO1K/C-S8c1 plasmids including the chosen mutations. As a control, a non-mutated 3242-bp NgoMIV segment derived from the original p3242/C-S8c1 plasmid was subjected to the same steps. The entire subcloned 3242-bp segment of a subset of the pO1K/C-S8c1 mutant plasmids, of non-mutated pO1K/C-S8c1, and of a revertant plasmid (obtained by back-mutating Ala-3146 to the original residue) was sequenced. Again no second site mutations were found. Transcription of Viral RNA and Electroporation of Eukaryotic Cells—pO1K/C-S8c1 and the mutant plasmids were linearized by digestion with HpaI, purified, and dissolved in RNase-free water. Infectious FMDV RNA was transcribed from the linearized plasmids by using the Riboprobe in vitro transcription system (Promega). The mixture contained 40 mm Hepes, pH 7.7, 6 mm magnesium acetate, 2 mm spermidine, 10 mm dithiothreitol, 1 unit/μl RNasin, 2.5 mm each of ribonucleoside triphosphates, 25 ng/μl linearized plasmid DNA, and 0.5 unit/μl SP6 RNA polymerase and was incubated for 2 h at 37 °C. The RNA concentration was estimated by agarose gel electrophoresis. About 2–4 × 106 BHK-21 cells from 60 to 80% confluent monolayers were resuspended in 0.5 ml of electroporation buffer containing 21 mm Hepes, pH 7.05, 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 6 mm glucose, and transferred to a Gene Pulser Cuvette (Bio-Rad) with a 0.4-cm electrode gap. FMDV RNA (3 μg) was added and electroporated using a Bio-Rad Gene Pulser set at 280 V, 250 microfarads, 400 ohm (two pulses). The cells were plated in a P60 Petri dish containing 4 ml of Dulbecco's modified Eagle's medium + 10% fetal calf serum and incubated at 37 °C. At 4 h post-transfection the medium was discarded, and 2 ml of fresh Dulbecco's modified Eagle's medium + 2% fetal calf serum was added. The incubation was continued for up to 90 h, with aliquots removed at specified intervals. The progeny virus suspension was clarified by centrifugation, and aliquots were stored at –70 °C. For nearly every transfection experiment, RNA was used immediately after transcription. Use of the same RNA either freshly prepared or frozen and thawed yielded very similar results. The same amount of RNA was used for every mutant. In each experiment the same amount of parent FMDV RNA, and no RNA, was used as a control. Immunofluorescence Assays—BHK-21 cells were electroporated with viral RNA as described above and subsequently cultured on coverslips. These were washed, incubated with 4% paraformaldehyde, washed again, and incubated with 10 mm glycine buffer, pH 8.5. The cell membranes were permeabilized by incubation with 0.2% Triton X-100 in phosphate-buffered saline (PBS). The coverslips were washed again, incubated in PBS + 3% bovine serum albumin (BSA), and then with monoclonal antibody (mAb) 5C4 (45Mateu M.G. Hernández J. Martínez M.A. Feigelstock D. Lea S. Pérez J.J. Giralt D. Stuart D. Palma E.L. Domingo E. J. Virol. 1994; 68: 1407-1417Crossref PubMed Google Scholar) (ascitic fluid diluted 1:500 in PBS + 3% BSA), washed thoroughly, incubated with secondary antibody (Alexa-488 from Molecular Probes, diluted 1:500 in PBS + 3% BSA), washed, mounted, and visualized in a fluorescence microscope. Titration and Amplification of Viruses and Extraction of Viral RNA—Virus titers were determined in plaque assays. Mutant virions obtained at very low titers were amplified by a single passage in BHK-21 cell monolayers at the highest possible multiplicity of infection. The progeny virus suspension was collected immediately after the cytopathic effect was observed, clarified, aliquoted, and frozen at –70 °C. RNA derived from virions collected from the supernatants of transfected or infected cell cultures was extracted using Trizol (Invitrogen) and precipitated with ethanol. The RNA was reverse-transcribed to DNA and amplified by reverse transcriptase-PCR as described previously (46Ruiz-Jarabo C.M. Arias A. Baranowski E. Escarmís C. Domingo E. J. Virol. 2000; 74: 3543-3547Crossref PubMed Scopus (162) Google Scholar), and the relevant segments were sequenced. Radioactive Labeling and Purification of Virions—FMDV virions were metabolically labeled with [35S]methionine (Redivue Pro-mix, Amersham Biosciences) and purified by sedimentation through a sucrose cushion followed by sucrose gradient centrifugation, essentially as described (47Díez J. Dávila M. Escarmís C. Mateu M.G. Domínguez J.J. Pérez E. Giralt E. Melero J.A. Domingo E. J. Virol. 1990; 64: 5519-5528Crossref PubMed Google Scholar). The fractions containing full virions (sedimentation coefficient 140 S) were extensively dialyzed against PBS and clarified by centrifugation. The purified virions were free of contaminants, as judged by overloaded SDS-urea-PAGE and Coomassie Blue staining. Capsid Dissociation Assays and Measurement of Rate Constants— Aliquots (0.3 ml) of 35S-labeled, purified virus were incubated at a constant temperature for different amounts of time, transferred to ice, loaded in 10–30% sucrose density gradients, and centrifuged at 4 °C in an SW40 rotor (Beckman Instruments) at 18,000 rpm for 18 h. The gradients were fractionated in 0.5-ml aliquots, and the radioactivity was determined using a liquid scintillation counter. In a second type of assay, virus suspensions obtained by transfection of infectious mutant RNA were diluted in Dulbecco's modified Eagle's medium + 2% fetal calf serum to a concentration of about 1000 plaque-forming units/ml. 100-μl aliquots in thin walled PCR tubes were incubated at a given temperature (42 °C unless indicated otherwise) for different amounts of time, and the remaining virus titers were determined in plaque assays. Non-mutated C-S8c1 virus obtained in parallel transfection experiments was used in each heat-inactivation experiment as a positive control. The experimental data were fitted to a first-order exponential decay by using the program Kaleidagraph (Abelbeck Software) and Equation 1, C=C0exp(-kt)(Eq. 1) where C is the virus titer at time t; C 0 is the virus titer at t = 0; and k is the dissociation rate constant. Structural Analyses of Capsid Subunit Interfaces—A silicon graphics work station and the Protein Data Bank coordinates for FMDVs of serotypes C (38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and O (37Acharya R. Fry E. Stuart D. Fox G. Rowlands D. Brown F. Nature. 1989; 337: 709-716Crossref PubMed Scopus (673) Google Scholar, 39Lea S. Abu-Ghazaleh R. Blakemore W. Curry S. Fry E. Jackson T. King A. Logan D. Newman J. Stuart D. Structure. 1995; 3: 571-580Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) were used. The crystallographic models were analyzed using the programs InsightII (Biosym Technologies) and RasMol (48Sayle R.A. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374-376Abstract Full Text PDF PubMed Scopus (2320) Google Scholar). Contact and solvent accessibility and modeling of mutations were done with the program Whatif (49Vriend G. J. Mol. Graphics. 1990; 8: 52-56Crossref PubMed Scopus (3369) Google Scholar) using the coordinates of all possible pairs of contacting subunits with different symmetry within the capsid. Analysis of Interpentameric Interactions within the FMDV Capsid—In the FMDV capsid, interpentamer interfaces involve essentially VP2-VP3 and VP2-VP2 pairwise contacts (37Acharya R. Fry E. Stuart D. Fox G. Rowlands D. Brown F. Nature. 1989; 337: 709-716Crossref PubMed Scopus (673) Google Scholar, 38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 39Lea S. Abu-Ghazaleh R. Blakemore W. Curry S. Fry E. Jackson T. King A. Logan D. Newman J. Stuart D. Structure. 1995; 3: 571-580Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 40Curry S. Fry E. Blakemore W. Abu-Ghazaleh R. Jackson T. King A. Lea S. Newman J. Rowlands D. Stuart D. Structure. 1996; 4: 135-145Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 41Fry E.E. Lea S.M. Jackson T. Newman J.W. Ellard F.M. Blakemore W.E. Abu-Ghazaleh R. Samuel A. King A.M. Stuart D.I. EMBO J. 1999; 18: 543-554Crossref PubMed Scopus (278) Google Scholar) (Fig. 1). In addition, the N termini of three symmetry-related VP2 from different pentamers form a short β-annulus around each 3-fold axis (38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The low electronic density associated with this annulus prevented the identification of the few side chains of this element, if any, involved in interactions. The structure of homologous FMDV isolates of three different serotypes, O1BFS (37Acharya R. Fry E. Stuart D. Fox G. Rowlands D. Brown F. Nature. 1989; 337: 709-716Crossref PubMed Scopus (673) Google Scholar, 41Fry E.E. Lea S.M. Jackson T. Newman J.W. Ellard F.M. Blakemore W.E. Abu-Ghazaleh R. Samuel A. King A.M. Stuart D.I. EMBO J. 1999; 18: 543-554Crossref PubMed Scopus (278) Google Scholar), which has been solved to very high crystallographic resolution (1.9 Å), C-S8c1 (38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and A22 (40Curry S. Fry E. Blakemore W. Abu-Ghazaleh R. Jackson T. King A. Lea S. Newman J. Rowlands D. Stuart D. Structure. 1996; 4: 135-145Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), revealed high conformational similarity and many conserved interfacial residues and interactions and allowed meaningful contact analyses in the structure of our model FMDV virus (C-S8c1) (38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Within the cut-off distances imposed (Table I), a total of 61 residues per capsid protomer appear involved in direct interpentamer interactions. The side chains of 42 of those residues are involved, beyond Cβ, in such interactions and constitute the interpentamer interface accessible to mutation (Table I and Fig. 1B). Eighteen of these side chains (only 8 being nonpolar) participate exclusively in van der Waals interactions, with an average of 2.8 contacts per residue. Very few (0–2) of these are carbon-carbon contacts (the only exceptions being residues Tyr-2200, Gln-3071, and Thr-2022 with 11, 5, and 3 carbon-carbon contacts, respectively). The remaining 24 polar side chains per protomer at the interfaces are involved in many interpentamer polar interactions. These include, per protomer subunit, a total of 18 different side chain-main chain or side chain-side chain hydrogen bonds, 2 strict salt bridges, 6 medium range charge-charge interactions, and a short range charge-helix dipole interaction (Table I). Most of these 24 side chains participate also in a variable but generally small numbers of van der Waals contacts.Table ISide chains involved in non-covalent interactions between pentamer subunits in the FMDV C-S8c1 capsidResidueaFor each residue, the first digit indicates the protein (either VP2 or VP3) and the last three digits the amino acid position according to Ref. 38.Charge-charge interactionsHydrogen bondsvan der Waals contactsE20111(K3118, 3.8 Å)7I20141R20182mc(N1017,Y1018)4H20211(E2213, 4.1 Å)5T20223T20231S20241sc(R3120)T20261sc(R3120)3Q20273mc(L3151,2xN3152)20L20512T20531sc(Q2057)Q20571sc(T2053)1R20601(E2213)1sc(E2213), 2mc (H3141,C3142)3K20631(E3146)2sc(E3146,H3144)12K20881(CtE2218, 5.5 Å)2K20961(CtE2218, 5.8 Å)Y20981E21081(R3120, 4.8 Å)T21101sc(R3120)1V21122N21144Q21154F21161M21543K21981(D3069, 4.8 Å)Y220012N22021E22132(R2060, H2021, 4.1 Å)1sc(R2060)5D30691(K2198)Q30717K31181(E2011, 3.8 Å)1mc(Q2115)7R31201(E2108, 4.8 Å)1mc(T2025), 3sc (S2024,T2026,T2110)3M31222H31411(helix dipole)6H31441sc(K2063)9E31461(K2063)1sc(K2063)4D31481mc(Q2027)7L31511N31521I31891T31905K31931mc(A2192)8a For each residue, the first digit indicates the protein (either VP2 or VP3) and the last three digits the amino acid position according to Ref. 38Lea S. Hernández J. Blakemore W. Brocchi E. Curry S. Domingo E. Fry E. Abu-Ghazaleh R. King A. Newman J. Stuart D. Mateu M.G. Structure. 1994; 2: 123-139Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar. Open table in a new tab In summary, apart from a limited number of main chain-main chain interactions, essentially all of the direct interpentamer interactions in the FMDV C-S8c1 capsid could be eliminated by truncation to alanine of 42 side chains per protomer that define the pentamer interfaces and that are involved mainly in polar interactions. Effects of the Truncation of the Side Chains Involved in Interpentameric Interactions on FMDV Infectivity—Each of the 42 interfacial side chains found involved in interpentameric interactions in FMDV C-S8c1 (Table I) was individually truncated to Ala to analyze first its individual role on the completion of the viral
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