The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26 (original) (raw)
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Journal of …, 1997
VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T61؍ icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of -sheets (ϳ80%), with a small ␣-helical component (ϳ15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of ϳ2 ؋ 10 ؊5 M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.
Journal of Virology, 2012
Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (ϳ100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.
Virology, 1997
The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T Å 16 icosahedral lattice. The major capsid protein, VP5 (MW Å 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.
Virology, 1997
The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T Å 16 icosahedral lattice. The major capsid protein, VP5 (MW Å 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.
Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions
Journal of virology, 1999
Herpes simplex virus type 1 virions were examined by electron cryomicroscopy, allowing the three-dimensional structure of the infectious particle to be visualized for the first time. The capsid shell is identical to that of B-capsids purified from the host cell nucleus, with the exception of the penton channel, which is closed. The double-stranded DNA genome is organized as regularly spaced ( approximately 26 A) concentric layers inside the capsid. This pattern suggests a spool model for DNA packaging, similar to that for some bacteriophages. The bulk of the tegument is not icosahedrally ordered. However, a small portion appears as filamentous structures around the pentons, interacting extensively with the capsid. Their locations and interactions suggest possible roles for the tegument proteins in regulating DNA transport through the penton channel and binding to cellular transport proteins during viral infection.
Journal of General Virology, 2005
Herpes simplex virus (HSV) capsids assemble, mature and package their viral genome in the nucleoplasm. They then exit the nucleus into the cytoplasm, where they acquire their final tegument and envelope. The molecular mechanism of cytoplasmic envelopment is unclear, but evidence suggests that the viral glycoprotein tails play an important role in the recruitment of tegument and capsids at the final envelopment site. However, due to redundancy in protein-protein interactions among the viral glycoproteins, genetic analysis of the role of individual glycoproteins in assembly has been difficult. To overcome this problem, a glutathione S-transferase fusion protein-binding assay was used in this study to test the interaction between the cytoplasmic tail of one specific viral glycoprotein, gD, and tegument proteins. The study demonstrated that the 38 kDa tegument protein VP22 bound specifically to the gD tail. This association was dependent on arginine and lysine residues at positions 5 and 6 in the gD tail. In addition, HSV-1 capsids bound the gD tail and exhibited a similar sequence dependence. It is concluded that VP22 may serve as a linker protein, mediating the interaction of the HSV capsid with gD.
Journal of Molecular Biology, 2013
The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM), and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A-and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein (GFP) was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9 Ångstrom resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.
Virology, 2007
The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The above results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.
Virology, 2009
Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least a portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.
Journal of General Virology, 1996
Herpes simplex virus type 1 (HSV-1) capsid assembly takes place in the nucleus of infected cells. However, when each of the outer capsid shell proteins, VP5, VP23 and VP26, is expressed in the absence of any other HSV-1 proteins, it does not localize to the nucleus but is distributed throughout the cell. We have previously shown that the HSV-I capsid scaffolding protein, preVP22a, can relocate VP5 into the nucleus but does not influence the distribution of VP23. We now demonstrate that the outer capsid shell protein, VP19C, is able to relocate both VP5 and VP23 separately into the nucleus. However, nuclear localization of VP26 is only observed when VP5 is present together with either VP19C or preVP22a. Thus, pair-wise interactions involving all of the abundant capsid proteins have now been identified. Electron microscope examination of insect cells coinfected with recombinant baculoviruses expressing VP19C and VP5 reveals the presence of 70nm diameter 'capsid-like' structures, suggesting that these two proteins can form the basic capsid shell.