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PubMed1998

Assaying for structural variation in the parvovirus capsid and its role in infection.

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.

Animals↗
PubMed2001

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Animals↗
PubMed1999

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

Bromovirus↗
PubMed1995

The 25 amino acid residues at the carboxy terminus of the herpes simplex virus type 1 UL26.5 protein are required for the formation of the capsid shell around the scaffold.

Herpes simplex virus type 1 (HSV-1) polypeptides specified by overlapping genes UL26 and UL26.5 form a scaffold around which the icosahedral capsid shell is assembled. In a series of cleavage events catalysed by the UL26-encoded protease, the full-length UL26 product is processed into capsid proteins VP24 and VP21 and the UL26.5 protein is converted into the capsid protein VP22a by the loss of 25 amino acids from its carboxy terminus. The roles of the UL26 and UL26.5 products were investigated using the baculovirus expression system, focusing on the function of the 25 residues cleaved from the UL26.5 protein. A key conclusion from electron microscopic analysis and protein expression studies is that the 25 amino acids at the carboxy terminus of the full-length UL26.5 protein are required for the interaction of the capsid shell proteins with the scaffold in the formation of intermediate capsids. When cells were multiply infected with baculoviruses expressing a truncated form of the UL26.5 product corresponding to VP22a and the essential components of the capsid shell, no capsids were detected, whereas large numbers of capsids were observed when the full-length UL26.5 product was used as a scaffold. The results are consistent with the proposal that cleavage of the UL26.5 product occurs after capsid assembly or when the UL26.5 protein is in a complex with one or more capsid shell proteins. Expression of VP22a in the absence or presence of capsid shell proteins resulted in the formation of large numbers of 60 nm scaffold-like particles. Since VP22a expressed from baculovirus was unable to participate in capsid assembly, these particles cannot be intermediates in the capsid assembly pathway but may be similar in structure to the protein cores present in HSV-1 immature (B) capsids.

Amino Acid Sequence↗
PubMed1995

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids.

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.

Anilino Naphthalenesulfonates↗
PubMed2003

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication.

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.

Amino Acid Sequence↗
PubMed1998

Herpes simplex virus DNA cleavage and packaging: association of multiple forms of U(L)15-encoded proteins with B capsids requires at least the U(L)6, U(L)17, and U(L)28 genes.

The U(L)15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U(L)15-encoded protein recognized three proteins with apparent Mrs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-Mr protein was detected in type C capsids and comigrated with the product of a U(L)15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-Mr proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(deltaU(L)15), a virus lacking an intact U(L)15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3' end of U(L)15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent Mrs of 83,000, 80,000, and 79,000 are products of U(L)15 with identical C termini. The 79,000-, 80,000-, and 83,000-Mr proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U(L)15-encoded proteins are integral components of B capsids. Only the 83,000-Mr protein was detected in B capsids purified from cells infected with viruses lacking the U(L)6, U(L)17, or U(L)28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-Mr proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-Mr U(L)15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.

Animals↗
PubMed1994

Mutations in the poliovirus P1 capsid precursor at arginine residues VP4-ARG34, VP3-ARG223, and VP1-ARG129 affect virus assembly and encapsidation of genomic RNA.

To begin to identify poliovirus capsid protein determinants required for assembly and RNA encapsidation, we have addressed the functional significance of three arginine residues of the poliovirus capsid in virus assembly and encapsidation of genomic RNA. These studies were conducted by using a recently described system in which recombinant vaccinia viruses are used to supply poliovirus capsid proteins in trans to a poliovirus subgenomic replicon [D. C. Ansardi, D. C. Porter, and C. D. Morrow (1993) J. Virol. 67, 3684-3690]. Two of the arginine residues, located at position 34 of VP4 (VP4-R034) and position 129 of VP1 (VP1-R129), are located within a cavity on the poliovirus capsid interior, whereas the third arginine, residue 223 of VP3 (VP3-R223), is located at a promoter-protomer interface. Five mutants were constructed by site-directed mutagenesis of poliovirus P1 capsid precursor cDNA to separately encode lysine or glutamine substitutions at VP4-R034 (VP4-R034K, VP4-R034Q), lysine or glutamine substitutions at residue 129 of VP1 (VP1-R129K, VP1-R129Q), or a lysine substitution at residue 223 of VP3 (VP3-R223K). Processed capsid proteins derived from the VP3-R223K, VP1-R129K, and VP1-R129Q mutant precursors were unstable and failed to assemble subviral particles or virions at 37 degrees. The assembly defect for cleavage products of the VP3-R223K precursor was partially overcome at 33 degrees, as empty capsids, but not mature virions, assembled from the mutant capsid subunits at the lower temperature. With regard to the third arginine residue analyzed, VP4-R034, processed capsid proteins derived from both the VP4-R034K and the VP4-R034Q mutant precursors assembled 155S virions at 37 degrees; however, capsid proteins derived from the VP4-R034Q precursor were temperature-sensitive for virion formation at 39.5 degrees. The reduced virion formation at 39.5 degrees was apparently a reflection of a defect in forming assembly competent subunits which also prevented accumulation of surplus VP4-R034Q subunits as empty capsids. By using graphics to display the poliovirus three-dimensional structure, the locations of these residues on the poliovirus capsid interior and their interactions with adjacent amino acids were visualized to provide structural explanations for the observed assembly defects which highlight the important role these residues play in capsid assembly and RNA encapsidation.

Arginine↗
PubMed2002

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids.

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.

Capsid Proteins↗
PubMed2001

Empty capsids in column-purified recombinant adenovirus preparations.

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

Adenoviridae↗
PubMed1994

Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses.

Immature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence of VP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.

Animals↗
PubMed1982

Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid.

It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA. Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentation, velocity sedimentation, and solid support-free electrophoresis, but was distinguished from capsid II by its apparent diversity during electrophoresis in agarose gels. Treatment of AG particles with trypsin converted most of them to particles that comigrated with trypsin-treated capsid II during electrophoresis in agarose gels. Irreversible binding of AG particles to agarose gels was shown to contribute to the apparent diversity of AG particles during agarose gel electrophoresis. The results of quantitation of AG particles and of capsid I and capsid II in lysates of a nonpermissive host infected with T7 amber mutants suggested that, in site of their capsid II-like properties, most AG particles were produced during assembly of capsid I and not during DNA packaging. The presence of AG particles in T7 lysates explains contradictions in previous data concerning the pathway of T7 assembly.

Capsid↗
PubMed2005

Extent of protein-protein interactions and quasi-equivalence in viral capsids.

Viral capsids are composed of multiple copies of one or a few gene products that self-assemble on their own or in the presence of the viral genome and/or auxiliary proteins into closed shells (capsids). We have analyzed 75 high-resolution virus capsid structures by calculating the average fraction of the solvent-accessible surface area of the coat protein subunits buried in the viral capsids. This fraction ranges from 0 to 1 and represents a normalized protein-protein interaction (PPI) index and is a measure of the extent of protein-protein interactions. The PPI indices were used to compare the extent of association of subunits among different capsids. We further examined the variation of the PPI indices as a function of the molecular weight of the coat protein subunit and the capsid diameter. Our results suggest that the PPI indices in T=1 and pseudo-T=3 capsids vary linearly with the molecular weight of the subunit and capsid size. This is in contrast to quasi-equivalent capsids with T>or=3, where the extent of protein-protein interactions is relatively independent of the subunit and capsid sizes. The striking outcome of this analysis is the distinctive clustering of the "T=2" capsids, which are distinguished by higher subunit molecular weights and a much lower degree of protein-protein interactions. Furthermore, the calculated residual (R(sym)) of the fraction buried surface areas of the structurally unique subunits in capsids with T>1 was used to calculate the quasi-equivalence of different subunit environments.

Binding Sites↗
PubMed1997

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles.

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Amino Acid Sequence↗
PubMed2005

Comparing capsid assembly of primate lentiviruses and hepatitis B virus using cell-free systems.

Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses. Capsid formation of these diverse viruses in a common eukaryotic extract was dependent on capsid protein concentration. HBV capsid assembly was also dependent on the presence of intact membrane surfaces. Surprisingly, not all of the primate lentiviral capsid proteins examined required myristoylation and intact membranes for assembly, even though all contain a myristoylation signal. These findings reveal significant diversity in how different capsid proteins assemble in the same cellular extract.

Amino Acid Sequence↗
PubMed1998

Herpes simplex virus type 1 cleavage and packaging proteins UL15 and UL28 are associated with B but not C capsids during packaging.

At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.

Animals↗
PubMed2004

The DNA cleavage and packaging protein encoded by the UL33 gene of herpes simplex virus 1 associates with capsids.

The U(L)33 gene of herpes simplex virus 1 (HSV-1) encodes a protein (pU(L)33) that is essential for the cleavage and packaging of concatameric herpesvirus DNA into preformed capsids. Previous data have suggested that the U(L)33 protein interacts with the cleavage and packaging proteins encoded by U(L)15 and U(L)28 that are known to associate with capsids. Examination of purified A capsids that lack DNA and are derived from aborted packaging events, B capsids that lack DNA, and C capsids that contain DNA revealed an association of the U(L)33 protein with all three capsid types. More U(L)33 protein was detected in A capsids than was present in B capsids. Capsid association was susceptible to guanidine-HCl treatment and independent of the presence of U(L)15 or U(L)28. Capsid association of pU(L)33 was also independent of U(L)6, which is believed to encode the portal into which DNA is inserted. These data suggest that pU(L)33 may act as part of the capsid-associated molecular machinery that translocates cleaved genomic DNA into the capsid interior.

Animals↗