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PubMed2003

Diagnostic potential of parechovirus capsid proteins.

To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione S-transferase (GST)-tagged recombinant proteins. The fusion proteins were used to raise antisera in rabbits. VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence. Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection. When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive. Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay. The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test. Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults. Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.

Adult↗
PubMed2006

Purified feline and canine transferrin receptors reveal complex interactions with the capsids of canine and feline parvoviruses that correspond to their host ranges.

The cell infection processes and host ranges of canine parvovirus (CPV) and feline panleukopenia virus (FPV) are controlled by their capsid interactions with the transferrin receptors (TfR) on their host cells. Here, we expressed the ectodomains of wild-type and mutant TfR and tested those for binding to purified viral capsids and showed that different naturally variant strains of the viruses were associated with variant interactions with the receptors which likely reflect the optimization of the viral infection processes in the different hosts. While all viruses bound the feline TfR, reflecting their tissue culture host ranges, a naturally variant mutant of CPV (represented by the CPV type-2b strain) that became the dominant virus worldwide in 1979 showed significantly lower levels of binding to the feline TfR. The canine TfR ectodomain did not bind to a detectable level in the in vitro assays, but this appears to reflect the naturally low affinity of that interaction, as only low levels of binding were seen when the receptor was expressed on mammalian cells; however, that was sufficient to allow endocytosis and infection. The apical domain of the canine TfR controls the specific interaction with CPV capsids, as a canine TfR mutant altering a glycosylation site in that domain bound FPV, CPV-2, and CPV-2b capsids efficiently. Enzymatic removal of the N-linked glycans did not allow FPV binding to the canine TfR, suggesting that the protein sequence difference is itself important. The purified feline TfR inhibited FPV and CPV-2 binding and infection of feline cells but not CPV-2b, indicating that the receptor binding may be able to prevent the attachment to the same receptor on cells.

Animals↗
PubMed2006

Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection.

Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.

Animals↗
PubMed2006

The African swine fever virus nonstructural protein pB602L is required for formation of the icosahedral capsid of the virus particle.

African swine fever virus (ASFV) protein pB602L has been described as a molecular chaperone for the correct folding of the major capsid protein p72. We have studied the function of protein pB602L during the viral assembly process by using a recombinant ASFV, vB602Li, which inducibly expresses the gene coding for this protein. We show that protein pB602L is a late nonstructural protein, which, in contrast with protein p72, is excluded from the viral factory. Repression of protein pB602L synthesis inhibits the proteolytic processing of the two viral polyproteins pp220 and pp62 and leads to a decrease in the levels of protein p72 and a delocalization of the capsid protein pE120R. As shown by electron microscopy analysis of cells infected with the recombinant virus vB602Li, the viral assembly process is severely altered in the absence of protein pB602L, with the generation of aberrant "zipper-like" structures instead of icosahedral virus particles. These "zipper-like" structures are similar to those found in cells infected under restrictive conditions with the recombinant virus vA72 inducibly expressing protein p72. Immunoelectron microscopy studies show that the abnormal forms generated in the absence of protein pB602L contain the inner envelope protein p17 and the two polyproteins but lack the capsid proteins p72 and pE120R. These findings indicate that protein pB602L is essential for the assembly of the icosahedral capsid of the virus particle.

African Swine Fever Virus↗
PubMed2006

Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments.

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.

Adenoviridae↗
PubMed2006

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions.

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

Animals↗
PubMed1976

Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.

Capsid↗
PubMed1977

Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits.

Cytoplasmic extracts of rhinovirus 1A-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts of the capsid peptides epsilon, alpha, and gamma, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only epsilon, alpha, and gamma chains in equal proportions, indicating that the 14S subunit is an oligomer of (epsilon gamma alpha) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion.

Capsid↗
PubMed1978

Head maturation pathway of bacteriophages T4 and T2. IV. In vitro transformation of T4 head-related particles produced by mutants in gene 17 to capsid-like structures.

T4 mutants in gene 17 accumulate particles which contain the main head protein in the cleaved form (gp23*) arranged in an unexpanded lattice (empty small particles), together with other expanded capsids (empty large particles). The isolated empty small particles can be transformed in vitro, by lowering the ionic strength, to capsid-like structures. This structural transformaton is not coupled to chemical modification of the structural proteins of the empty small particles. In contrast to unexpanded particles that are easily dissociated, the transformed structures are as resistant to dissociation as other T-even head-related particles with expanded lattice. Furthermore, the transformed particles are able to bind in vitro hoc and soc proteins, rendering capsids indistinguishable from the normal T4 capsids both morphologically and by their stability against denaturing agents. Our results indicate that the in vitro transformation of the empty small particles might mimic important and characteristic aspects of the in vivo maturation of T4 heads, thus suggesting a possible role of the "cleaved but unexpanded" particle in the maturation pathway of the T4 shell.

Capsid↗
PubMed1979

Differential adsorption of polyoma virions and capsids to mouse kidney cells and guinea pig erythrocytes.

Adsorption of 125I-labeled polyoma virions and capsids to the surface of mouse kidney cells (MKC) and guinea pig erythrocytes was examined. Purified polyoma capsids lack the ability to compete with polyoma virions for specific binding sites on the surface of MKC. These same capsids were, however, able to block virion adsorption to guinea pig erythrocytes. UV-inactivated virions blocked cellular receptors on MKC and thus inhibited infectious virions from infecting the cells. Capsids were unable to inhibit virion infection of MKC. Adsorption of polyoma virions to MKC and infection of these cells were found to be independent of the ability of the virions to agglutinate guinea pig erythrocytes.

Adsorption↗
PubMed1987

Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins.

We generated defined alterations in poliovirus protein-processing substrates and assayed the effects of these alterations with an in vitro expression system. A complete cDNA copy of the poliovirus genome was inserted into a bacteriophage T7 transcription vector. Using this expression template, we produced RNA transcripts containing defined regions of the poliovirus capsid precursor polypeptide (P1) and RNA transcripts containing mutations in the P1 and P2 regions. In vitro translation of P1-derived transcripts allowed us to characterize the 3C-mediated cleavage of P1 to capsid proteins. We demonstrated that, for either posttranslational or cotranslational cleavage at any of the Q-G amino acid pairs within P1, almost the entire P1 precursor is required. We also demonstrated that minimal sequences 3' to the 2A coding sequence are required to generate active 2A proteinase in vitro and that two specific four-amino-acid insertions in protein 2C do not alter 2A- or 3C-mediated processing of the poliovirus polyprotein. In addition, we demonstrated that substantial deletion of P1 sequences does not alter 2A-mediated cleavage of the Y-G site at the P1-P2 junction. These results allowed us to compare the P1 sequences required for 2A- versus 3C-mediated processing of the capsid precursor, and we discuss these results in the context of the three-dimensional structure of the capsid proteins.

Amino Acid Sequence↗
PubMed1987

Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids.

The role of myristylation, a fatty acid modification of nascent polypeptides, in the assembly and intracellular transport of D-type retroviral capsids was investigated through the use of oligonucleotide-directed mutagenesis. Myristic acid is normally esterified through an amide linkage to a glycine residue at the amino terminus of the Mason-Pfizer monkey virus gag gene products. Mutant pA-1, which has a codon for valine substituted for that of the normally myristylated glycine, is completely noninfectious. While the mutant gag polyprotein precursors are synthesized at normal levels, they are not myristylated and are not cleaved to the mature virion proteins. No extracellular virus particles are released from mutant pA-1-infected cells, but intracytoplasmic A-type particles (capsids) accumulate in the cytoplasm. Since none of the intracellular capsids can be found associated with the plasma membrane, these results strongly suggest that myristylation is a critical signal for intracytoplasmic transport of completed viral capsids to their normal site of budding and release.

Capsid↗
PubMed1992

Envelope glycoprotein and CD4 independence of vpu-facilitated human immunodeficiency virus type 1 capsid export.

The effect of vpu on the release of human immunodeficiency type 1 capsid proteins was examined in the presence or absence of virus-encoded envelope glycoproteins as well as in cells which constitutively express either the CD4 or CD8 protein. The results show that vpu-mediated facilitated export of capsid proteins from HeLa cells does not require expression of the envelope glycoprotein. The experiments also show that export of virus capsid proteins from HeLa cells facilitated by vpu is not affected by coexpression of either the CD4 or CD8 protein. The vpu protein acts in trans to facilitate export of virus capsid proteins from HeLa cells.

Antigens, CD↗
PubMed1992

RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication.

The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus.

The capsid protein of duck hepatitis B virus (DHBV) is phosphorylated at multiple sites during viral infection. A cluster of sites is located near the C terminus of the 262-amino-acid protein. We have used site-directed mutagenesis to show that three serines and one threonine serve as phosphate acceptor amino acids in the C terminus. An additional six potential phosphate acceptor sites in this region were apparently not utilized. Each serine or threonine that served as a phosphate acceptor was adjacent to a downstream proline, while all six serines that were not acceptors for phosphate residues lacked adjacent downstream prolines. Mutation of the downstream proline to glycine at each site had the same effect as mutating the serine itself, suggesting an SP or TP motif as an essential feature for capsid protein phosphorylation. Phosphorylation at these four sites resulted in complex shifts in electrophoretic mobility in sodium dodecyl sulfate gels of the capsid protein or of a C-terminal peptide containing the phosphorylated sites, suggesting that specific conformations of the C terminus are associated with different combinations of phosphorylated serines. We speculate that distinct functions of the C terminus may be associated with different phosphorylated domains on the intact capsid.

Amino Acid Sequence↗
PubMed1994

Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.

Animals↗
PubMed1995

The C-terminal 25 amino acids of the protease and its substrate ICP35 of herpes simplex virus type 1 are involved in the formation of sealed capsids.

The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.

Animals↗
PubMed1996

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.

Animals↗