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PubMed2006

Stochastic kinetics of viral capsid assembly based on detailed protein structures.

We present a generic computational framework for the simulation of viral capsid assembly which is quantitative and specific. Starting from PDB files containing atomic coordinates, the algorithm builds a coarse-grained description of protein oligomers based on graph rigidity. These reduced protein descriptions are used in an extended Gillespie algorithm to investigate the stochastic kinetics of the assembly process. The association rates are obtained from a diffusive Smoluchowski equation for rapid coagulation, modified to account for water shielding and protein structure. The dissociation rates are derived by interpreting the splitting of oligomers as a process of graph partitioning akin to the escape from a multidimensional well. This modular framework is quantitative yet computationally tractable, with a small number of physically motivated parameters. The methodology is illustrated using two different viruses which are shown to follow quantitatively different assembly pathways. We also show how in this model the quasi-stationary kinetics of assembly can be described as a Markovian cascading process, in which only a few intermediates and a small proportion of pathways are present. The observed pathways and intermediates can be related a posteriori to structural and energetic properties of the capsid oligomers.

Algorithms↗
PubMed2006

Dynamic pathways for viral capsid assembly.

We develop a class of models with which we simulate the assembly of particles into T1 capsidlike objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile; for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

Capsid↗
PubMed1998

Determination of encephalomyocarditis viral diabetogenicity by a putative binding site of the viral capsid protein.

The molecular mechanism by which some, but not all, variants of encephalomyocarditis (EMC) virus selectively infect pancreatic beta-cells in mice and induce IDDM has been an enigma for more than a decade. We report here that the binding site of the EMC viral capsid protein VP1 determines viral diabetogenicity. Recombinant chimeric EMC viruses containing threonine, serine, proline, aspartic acid, or valine at position 152 of the major capsid protein VP1 bind poorly to beta-cells. In contrast, recombinant chimeric EMC viruses containing alanine or glycine at position 152 of the VP1 bind efficiently to and infect beta-cells, resulting in the development of diabetes. Three-dimensional molecular modeling reveals that the van der Waals interactions are greater and the residues surrounding position 152 of the VP1 are more closely packed in recombinant chimeric viruses containing threonine, serine, proline, aspartic acid, or valine at position 152 than in recombinant chimeric viruses containing alanine or glycine at the same position. Our studies reveal that the surface areas surrounding alanine or glycine at position 152 of the VP1 are more accessible, thus increasing the availability of the binding sites for attachment to beta-cell receptors and resulting in viral infection and the development of diabetes.

Animals↗
PubMed2001

Effect of a valine residue at codon 352 of the VP2 capsid protein on in vivo replication and pathogenesis of Aleutian disease parvovirus in mink.

OBJECTIVE: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10. ANIMALS: 32 eight-month-old female sapphire mink (Mustela vison). PROCEDURE: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy. RESULTS: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V. CONCLUSIONS AND CLINICAL RELEVANCE: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.

Aleutian Mink Disease↗
PubMed1990

[Probable epitopes of adenovirus capsid proteins].

The hydrophilicity and flexibility patterns of two capsid proteins of type 2 human adenovirus (hexon and fiber) were obtained. Several sites with maximal hydrophilicity and flexibility were revealed; their correlation was partly established. The role of these sites in the formation of adenoviral capsid protein epitopes is discussed.

Adenoviruses, Human↗
PubMed1994

Structure of rotavirus particle: interaction of the inner capsid protein VP6 with the core polypeptide VP3.

The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able to reconstitute the particle with both the mutant cores and VP6. These results suggest the existence of various steps for the assembly of single shelled particles, where the VP6-VP3 interaction seems to be important for recovery of RNA polymerase activity.

Antibodies, Viral↗
PubMed2000

Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein.

A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats. Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses. Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation. Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 [Li et al. (1997) Journal of Medical Virology 52:289-300], a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients. Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition). Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection.

Adjuvants, Immunologic↗
PubMed1993

Epitope analysis of the T cell response to a complex antigen: proliferative responses to human rhinovirus capsids.

Understanding the factors which regulate the repertoire of a T cell response is important when selecting T helper cell epitopes for inclusion in synthetic viral vaccines. In this study we have examined the T cell response to human rhinovirus (HRV) type 1 A in a mouse model system, using a comprehensive set of synthetic peptides which span all four of the proteins which make up the HRV capsid. This constitutes the first study to use a set of peptides covering the entire sequence of all structural proteins of any virus. This study identifies the major proliferative (CD4) T cell epitopes within the minor receptor group HRV 1 A, and analyzes these epitopes with relation to their location within the three-dimensional structure of the virus. The proliferative response to HRV is highly selective, with strong responses to only a very small number of epitopes, many of which are grouped together within restricted areas of the primary structure of the HRV proteins. The repertoire of the response is almost entirely specific to the major histocompatibility complex haplotype of the host. The major T cell epitopes are spatially distinct from the sites of the major antibody recognition sites, and are buried within the viral capsid. In striking contrast to the antibody responses, the T cell responses are highly cross-reactive against a wide variety of viral serotypes.

Amino Acid Sequence↗
PubMed1989

Role of myristoylation of poliovirus capsid protein VP4 as determined by site-directed mutagenesis of its N-terminal sequence.

Mutations were introduced by oligonucleotide-directed mutagenesis into the cDNA of poliovirus type 1 (Mahoney) in the region coding for the first five amino acids (myristoylation signal) of the viral capsid protein precursor P1. The cDNAs were then transcribed in vitro and the properties of the transcripts carrying the mutations studied in vitro by translation in a reticulocyte lysate or in vivo upon transfection of primate cells. Mutation of amino acid residue number 5 (Ser5----Thr) did not affect the viral phenotype, whereas mutations of residues number 1 (Gly1----Arg), 2 (Ala2----Pro) or 5 (Ser5----Pro) prevented myristoylation of P1 and were lethal. However, delayed production of virus was occasionally observed as the result of reverse mutations, which were found to restore a functional myristoylation signal as well as a wild-type virus phenotype. Thus, the myristoylation signal of the poliovirus polyprotein can accommodate Ala, Ser, Thr or Leu residues at position 2 and Ser, Thr or Ala residues at position 5. Mutations that altered myristoylation of P1 and affected virus viability did not prevent replication of the viral RNA but severely impeded in vitro processing of P1. This suggests that myristoylation plays a role in poliovirus capsid protein assembly.

Amino Acid Sequence↗
PubMed1983

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus.

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.

Animals↗
PubMed1994

Avidities of IgG directed against viral capsid antigen or early antigen: useful markers for significant Epstein-Barr virus serology.

Classical Epstein-Barr virus (EBV) serology can be misleading in some cases due to the variability of the viral capsid antigen (VCA)-IgM response, persistent or reactivated VCA-IgM, or loss of anti-EBNA-1 during suppression of the cellular immune system. Therefore, we studied the usefulness and significance of avidity determinations of VCA-IgG and EA-IgG to achieve unequivocal interpretation of serological results. Avidities of EBV capsid antigen-specific IgG (VCA-IgG) and early antigen-specific IgG (EA-IgG) were determined by indirect immunofluorescence during and after acute EBV infection. Low-avidity antibodies were removed from antigen-antibody complexes by incubation with 6 M urea for 3 minutes. The analysis of 105 sera taken at defined time spans with regard to the onset of clinical symptoms allowed us to determine the kinetics of maturation of avidity of VCA-IgG. All sera had low-avidity antibodies at the onset of disease. More than 90% of the sera showed an avidity index below 0.25 during the first 10 days after the onset of disease. Fifty percent of the sera exhibited an avidity index of 0.25 or above 20-30 days after the onset of clinical symptoms. Sera from past infections uniformly exhibited avidity indices of 0.5 or 1. Avidity of EA-IgG may still be low when avidity of VCA-IgG is already borderline or high, thus allowing further differentiation of acute and recent infections. Avidity determination represents an important additional marker of serology in classical cases and allows diagnosis in aberrant cases, such as acute infections with low or undetectable VCA-IgM.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed2001

In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines.

Core assembly, a key step in the retroviral life cycle, is poorly understood. Previous studies have shown that the entire gag region is needed to form the assembled particles. In this report, we have shown that the assembly process is driven by recombinant capsid protein (p26) of feline immunodeficiency virus itself. Proteins are expressed in a bacterial system and soluble forms of wild-type and modified proteins are purified from bacterial extracts and are examined on gel-filtration chromatography fitted to an HPLC system. It has also been shown that changing residue Cys190 (one of the two conserved cysteines of feline immunodeficiency virus which are also conserved for all the immunodeficiency viruses including HIV) to serine by site-directed mutagenesis disrupts the assembly process. In addition, this modification causes considerable thermal instability of the protein while substitutions at nonconserved cysteines do not significantly affect the thermal stability and assembly of the protein. These findings indicate that conserved cysteine residues play a vital role in the capsid protein assembly and, therefore, are critical for virus infectivity.

Amino Acid Sequence↗
PubMed1994

Crystal structure of bacteriophage fr capsids at 3.5 A resolution.

The structure of recombinant capsids of the bacterial virus fr has been determined by X-ray crystallography at 3.5 A resolution. The capsids were produced by expressing the fr coat protein in Escherichia coli, the natural host of the virus, and are probably essentially identical to the protein shell of the native virus. The structure was determined using molecular replacement with the protein shell of the related MS2 virus, and refined to a crystallographic R-factor of 0.228. A comparison of the protein shells of the viruses shows that they are very similar, and indicates that they may have a similar regulation of the assembly of the quasi-symmetrical protein shell.

Amino Acid Sequence↗
PubMed1997

Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway.

The different partially folded states of the capsid protein that appear in the disassembly pathway of cowpea severe mosaic virus (CPSMV) were investigated by examining the effects of hydrostatic pressure, sub-zero temperatures and urea. The conformational states of the coat protein were analyzed by their intrinsic fluorescence, binding of bis(8-anilinonaphthalene-1-sulfonate) (bis-ANS) and susceptibility to trypsin digestion. CPSMV could be disassembled by pressure at 2.5 kbar. Intrinsic fluorescence and hydrodynamic measurements showed that pressure-induced dissociation was completely reversible. Virus pressurization in the presence of ribonuclease revealed that viral RNA was not exposed, since it was not digested by the enzyme, suggesting the maintenance of protein-nucleic acid interactions under pressure. When the temperature was decreased to -10 degrees C under pressure, CPSMV disassembly became an irreversible process and in this condition, viral RNA was completely digested by ribonuclease. These results suggest a relationship between protein-RNA interactions and CPSMV assembly. Bis-ANS binding and trypsin digestion of coat proteins revealed that they assume a different conformation when they are denatured by low temperatures under pressure or than when they are denatured by urea at atmospheric pressure. The results indicate that the coat proteins can exist in at least four states: (1) The native conformation in the virus capsid; (2) bound to RNA when the virus is dissociated by pressure at room temperature, assuming a conformation that retains the information for reassembly; (3) free subunits in a molten-globule conformation when the virus is dissociated by low temperature under pressure; and (4) free subunits completely unfolded by high concentrations of urea.

Anilino Naphthalenesulfonates↗
PubMed2000

Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses.

Canine parvovirus (CPV) emerged in 1978 as a host range variant of feline panleukopenia virus (FPV). This change of host was mediated by the mutation of five residues on the surface of the capsid. CPV and FPV enter cells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range and tissue specificity are largely determined by events occurring after cell entry. We have determined the structures of a variety of strains of CPV and FPV at various pH values and in the presence or absence of Ca(2+). The largest structural difference was found to occur in a flexible surface loop, consisting of residues 359 to 375 of the capsid protein. This loop binds a divalent calcium ion in FPV and is adjacent to a double Ca(2+)-binding site, both in CPV and FPV. Residues within the loop and those associated with the double Ca(2+)-binding site were found to be essential for virus infectivity. The residues involved in the double Ca(2+)-binding site are conserved only in FPV and CPV. Our results show that the loop conformation and the associated Ca(2+)-binding are influenced by the Ca(2+) concentration, as well as pH. These changes are correlated with the ability of the virus to hemagglutinate erythrocytes. The co-localization of hemagglutinating activity and host range determinants on the virus surface implies that these properties may be functionally linked. We speculate that the flexible loop and surrounding regions are involved in binding an as yet unidentified host molecule and that this interaction influences host range.

Amino Acid Substitution↗
PubMed2001

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia.

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.

Aldehydes↗
PubMed2001

The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus.

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.

Amino Acid Sequence↗
PubMed2002

Analysis of Rous sarcoma virus capsid protein variants assembled on lipid monolayers.

During assembly and morphogenesis of Rous sarcoma virus (RSV), proteolytic processing of the structural precursor (Pr76Gag) protein generates three capsid (CA) protein variants, CA476, CA479, and CA488. The proteins share identical N-terminal domains (NTDs), but are truncated at residues corresponding to gag codons 476, 479, and 488 in their CA C-terminal domains (CTDs). To characterize oligomeric forms of the RSV CA variants, we examined 2D crystals of the capsid proteins, assembled on lipid monolayers. Using electron microscopy and image analysis approaches, the CA proteins were observed to organize in hexagonal (p6) arrangements, where rings of membrane-proximal NTD hexamers were spaced at 95 A intervals. Differences between the oligomeric structures of the CA variants were most evident in membrane-distal regions, where apparent CTDs interconnect hexamer rings. In this region, CA488 connections were observed readily, while CA476 and CA479 contacts were resolved poorly, suggesting that in vivo processing of CA488 to the shorter forms may permit virions to adopt a dissembly-competent conformation. In addition to crystalline arrays, the CA479 and CA488 proteins formed small spherical particles with diameters of 165-175 A. The spheres appear to be arranged from hexamer or hexamer plus pentamer ring subunits that are related to the 2D crystal forms. Our results implicate RSV CA hexamer rings as basic elements in the assembly of RSV virus cores.

Amino Acid Sequence↗