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At least 109 records · Page 6Linked to original sources
PubMed1995

Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability.

Bacteriophage P22 is a double-stranded DNA containing phage. Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid. The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously [Prevelige, P. E., King, J., & Silva, J. L. (1994) Biophys. J. 66, 1631-1641]. The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar. However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized. We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods. While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 degrees C), the G232D mutant shells showed time-dependent dissociation under these conditions. Decreasing the temperature to 1 degree C dramatically accelerated the dissociation of G232D mutant under applied pressure. On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant. The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage P22↗
PubMed1994

Expression of the beet yellows closterovirus capsid protein and p24, a capsid protein homologue, in vitro and in vivo.

The positive-sense RNA genome of beet yellows closterovirus (BYV) encompasses open reading frames (ORFs) for the viral capsid protein (CP, ORF 6) and for a CP homologue (p24, ORF 5). The sequences of the ORFs 5 and 6 were inserted into an Escherichia coli expression vector, pQE-9, under the control of the bacteriophage T5 promoter. The proteins were expressed in bacteria, purified, and used for antiserum production in rabbits. The recombinant BYV CP and p24 showed serological cross-reactions when probed with each antiserum on Western blots. The cross-reactions of the anti-p24 serum with the CP, and of the anti-CP serum with the p24, were abolished by preadsorption with the heterologous antigens, suggesting that CP and p24 share a common epitope(s) resistant to SDS denaturation. Cross-reactivity of the soluble CP and p24 was also observed in indirect plate-trapped antigen ELISA, whereas virtually none was encountered in double-antibody sandwich ELISA. Using a polyclonal anti-p24 serum preadsorbed with the recombinant CP, the p24 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected Tetragonia expansa indicated that both proteins are predominantly located in the soluble fraction of the host cells. Primer extension analysis of the individual double-stranded forms of the subgenomic RNAs bearing the CP and p24 genes allowed them to be mapped and their 5' start sites to be located at nucleotide positions 13,588 and 12,815, respectively, in the complete genome sequence. This corresponds to the 5' untranslated regions of 52 and 105 nucleotides in the subgenomic RNAs for CP and p24, respectively. The data obtained indicate that the synthesis of both subgenomic RNAs is initiated on a negative RNA strand at an adenosine residue found within the conserved sequence 5' CCAUUUPyA (shown as positive-sense), which may thus represent a core element of the subgenomic promoter. This conserved sequence also resembles the sequences at the 5' ends of the CP subgenomic RNAs of tobamoviruses and the Bromoviridae family members, the viruses evolutionarily most closely related to BYV.

Antigens, Viral↗
PubMed2003

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes.

The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

African Horse Sickness↗
PubMed1997

Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.

Amino Acid Sequence↗
PubMed2003

Human immunodeficiency virus type 1 N-terminal capsid mutants containing cores with abnormally high levels of capsid protein and virtually no reverse transcriptase.

We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

Capsid Proteins↗
PubMed1991

Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2.

The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of bluetongue virus (BTV) United States serotype 13 was determined. The comparison of the predicted amino acid sequence of the encoded protein (VP5) with the sequences of VP5 from two BTV United States serotypes, 10 and 2, and two isolates of BTV serotype 1 (Australian and South African), revealed that the protein is highly conserved among the different serotypes despite their geographical separation.

Amino Acid Sequence↗
PubMed1992

Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles.

A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.

Aleutian Mink Disease↗
PubMed1978

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.

Agarose gel electrophoresis of the following was performed in 0.05 M sodium phosphate-0.001 M MgCl2 (pH 7.4): (i) bacteriophage T7; (ii) a T7 precursor capsid (capsid I), isolated from T7-infected Escherichia coli, which has a thicker and less angular envelope than bacteriophage T7; (iii) a second capsid (capsid II), isolated from T7-infected E. coli, which has a bacteriophage-like envelope; and (iv) capsids (capsid IV) produced by temperature shock of bacteriophage T7. Bacteriophage T7 and all of the above capsids migrated towards the anode. In a 0.9% agarose gel, capsid I had an electrophoretic mobility of 9.1 +/- 0.4 X 10(-5) cm2/V.s; bacteriophage T7 migrated 0.31 +/- 0.02 times as fast as capsid I. The mobilities of different preparations of capsid II varied in such gels: the fastest-migrating capsid II preparation was 0.51 +/- 0.03 times as fast as capsid I and the slowest was 0.37 +/- 0.02 times as fast as capsid I. Capsid IV with and without the phage tail migrated 0.29 +/- 0.02 and 0.42 +/- 0.02 times as fast as capsid I. The results of the extrapolation of bacteriophage and capsid mobilities to 0% agarose concentration indicated that the above differences in mobility are caused by differences in average surface charge density. To increase the accuracy of mobility comparisons and to increase the number of samples that could be simultaneously analyzed, multisample horizontal slab gels were used. Treatment with the ionic detergent sodium dodecyl sulfate converted capsid I to a capsid that migated in the capsid II region during electrophoresis through agarose gels. In the electron microscope, most of the envelopes of these latter capsids resembled the capsid II envelope, but some envelope regions were thicker than the capsid II envelope.

Capsid↗
PubMed2003

Residues of VP26 of herpes simplex virus type 1 that are required for its interaction with capsids.

VP26 is the smallest capsid protein and decorates the outer surface of the capsid shell of herpes simplex virus. It is located on the hexons at equimolar amounts with VP5. Its small size (112 amino acids) and high copy number make it an attractive molecule to use as a probe to investigate the complex pattern of capsid protein interactions. An in vitro capsid binding assay and a green fluorescent protein (GFP) localization assay were used to identify VP26 residues important for its interaction with capsids. To test for regions of VP26 that may be essential for binding to capsids, three small in-frame deletion mutations were generated in VP26, Delta18-25, Delta54-60, and Delta93-100. Their designations refer to the amino acids deleted by the mutation. The mutation at the C terminus of the molecule, which encompasses a region of highly conserved residues, abolished binding to the capsid and the localization of GFP to the nucleus in characteristic large puncta. Additional mutations revealed that a region of VP26 spanning from residue 50 to 112 was sufficient for the localization of the fused protein (VP26-GFP) to the nucleus and for it to bind to capsids. Using site-directed mutagenesis of conserved residues in VP26, two key residues for protein-protein interaction, F79 and G93, were identified as judged by the localization of GFP to nuclear puncta. When these mutations were analyzed in the capsid binding assay, they were also found to eliminate binding of VP26 to the capsid structure. Surprisingly, additional mutations that affected the ability of VP26 to bind to capsids in vitro were uncovered. Mutations at residues A58 and L64 resulted in a reduced ability of VP26 to bind to capsids. Mutation of the hydrophobic residues M78 and A80, which are adjacent to the hydrophobic residue F79, abolished VP26 capsid binding. In addition, the block of conserved amino acids in the carboxy end of the molecule had the most profound effect on the ability of VP26 to interact with capsids. Mutation of amino acid G93, L94, R95, R96, or T97 resulted in a greatly diminished ability of VP26 to bind capsids. Yet, all of these residues other than G93 were able to efficiently translocate or concentrate GFP into the nucleus, giving rise to the punctate fluorescence. Thus, the interaction of VP26 with the capsid appears to occur through at least two separate mechanisms. The initial interaction of VP26 and VP5 may occur in the cytoplasm or when VP5 is localized in the nucleus. Residues F79 and G93 are important for this bi-molecular interaction, resulting in the accumulation of VP26 in the nucleus in concentrated foci. Subsequent to this association, additional amino acids of VP26, including those in the C-terminal conserved domain, are important for interaction of VP26 with the three-dimensional capsid structure.

Amino Acid Sequence↗
PubMed1993

Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.

The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.

Capsid↗
PubMed2006

Herpes simplex virus type 1 DNA-packaging protein UL17 is required for efficient binding of UL25 to capsids.

Herpes simplex virus type 1 packages its DNA genome into a precursor capsid, referred to as the procapsid. Of the three capsid-associated DNA-packaging proteins, UL17, UL25, and UL6, only UL17 and UL6 appear to be components of the procapsid, with UL25 being added subsequently. To determine whether the association of UL17 or UL25 with capsids was dependent on the other two packaging proteins, B capsids, which lack viral DNA but retain the cleaved internal scaffold, were purified from nonpermissive cells infected with UL17, UL25, or UL6 null mutants and compared with wild-type (wt) B capsids. In the absence of UL17, the levels of UL25 in the mutant capsids were much lower than those in wt B capsids. These results suggest that UL17 is required for efficient incorporation of UL25 into B capsids. B capsids lacking UL25 contained about twofold-less UL17 than wt capsids, raising the possibilities that UL25 is important for stabilizing UL17 in capsids and that the two proteins interact in the capsid. The distribution of UL17 and UL25 on B capsids was examined using immunogold labeling. Both proteins appeared to bind to multiple sites on the capsid. The properties of the UL17 and UL25 proteins are consistent with the idea that the two proteins are important in stabilizing capsid-DNA structures rather than having a direct role in DNA packaging.

Animals↗
PubMed1995

Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly.

The production and biochemical and physiocochemical analysis are described of recombinant-produced hepatitis B virus core antigen (HBcAg capsid) and the corresponding particle produced by a deletion mutant missing the C-terminal 39 residues (HBeAg). Conditions for producing HBeAg from HBcAg capsids by in vitro proteolysis are also described. The morphology and masses of these capsids were determined by scanning transmission electron microscopy. Both HBcAg and HBeAg capsids comprise two size classes that correspond to icosahedral lattices with triangulation numbers (T) of 3 and 4, containing 180 and 240 subunits per capsid, respectively. This dimorphism was confirmed by sedimentation equilibrium and sedimentation velocity measurements on a Beckman Optima XL-A analytical ultracentrifuge. More than 60% of HBcAg capsids were T = 4, whereas only 15-20% of HBeAg capsids were of this size class: the remainder, in each case, were T = 3. Circular dichroism and Raman spectroscopy were used to determine the overall secondary structures of HBcAg and HBeAg capsids. Both have high alpha-helical contents, implying that this capsid protein does not conform to the canonical beta-barrel motif seen for all plant and animal icosahedral viral capsids solved to date. We suggest that the C-terminal domain of HBcAg has a random coil conformation. In vitro dissociation of HBeAg capsids under relatively mild conditions yielded stable dimers. The reassociation of HBeAg dimers into capsids appears to be driven by hydrophobic processes at neutral pH. Capsid assembly is accompanied by little change in subunit conformation as judged by circular dichroism and fluorescence spectroscopy. The thermal stability of HBcAg capsids was compared calorimetrically with that of in vitro assembled HBeAg capsids. Both have melting temperatures > 90 degrees C, implying that the C-terminal region makes little difference to the thermal stability of HBcAg: nevertheless, we discuss its possible role in facilitating disassembly and the release of viral nucleic acid.

Calorimetry, Differential Scanning↗
PubMed2005

Maturation of papillomavirus capsids.

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.

Animals↗
PubMedDate not supplied

BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly.

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.

Antiviral Agents↗