Identification of disulfide-linked protein complexes in the nucleocapsids of herpes simplex virus type 2

Zweig, M; Heilman, C J; Hampart, Berge

doi:10.1016/0042-6822(79)90474-4

Cited by 36 publications

(29 citation statements)

References 11 publications

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“…The observation that the portal ring uses disulfide bond formation for stability is interesting in light of previous observations that HSV capsids contain disulfide bonds potentially involving VP5, triplex, and scaffold proteins (43,48). We have confirmed the existence of disulfide bonds in capsids and virions and have demonstrated that these bonds contribute to overall capsid stability (38a).…”

Section: Discussionsupporting

confidence: 66%

“…UL6 portal rings from insect cells infected with recombinant baculovirus were disrupted when exposed to reducing agents. Although disulfide bonds have been reported previously between HSV-2 capsid proteins (48) and in HSV-1 scaffold pro-teins (43), this is the first report of disulfide linkages in the portal ring. The mutational analysis of UL6 identified cysteines 166 and 254 as essential for (i) intermolecular disulfide bond formation; (ii) the formation and/or stability of portal rings; and (iii) the production of procapsids that are capable of encapsidation.…”

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confidence: 71%

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Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

Albright

Nellissery

Szczepaniak

et al. 2011

J Virol

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The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868-8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.The products of herpes simplex virus 1 (HSV-1) DNA replication are head-to-tail concatemers which are resolved into monomeric genomic units and packaged into a preformed capsid shell in the nucleus of the infected cell (reviewed in references 2, 6, and 10). The HSV-1 capsid shell is composed of the major capsid protein (VP5), two triplex proteins (VP19C and VP23), and VP26. Minor capsid proteins include UL6, UL15, UL17, UL25, UL28, and UL33. The process of cleavage and DNA packaging requires the six minor capsid proteins as well as UL32, which is not found associated with capsids (2, 6, 10, 21).HSV capsid formation and genome encapsidation are reminiscent of the double-stranded DNA bacteriophages, in that a procapsid shell is preassembled around a scaffolding protein that is not present in the mature virion (3, 37, 38). Bacteriophage and herpesviruses share an important structural element, a dodecameric portal ring located at a unique capsid vertex (8,9,28,40). During HSV genome encapsidation, the portal ring provides a docking site for the terminase, an ATPdriven molecular motor that facilitates the uptake of viral DNA (34,42,45,46). Terminase is responsible not only for viral DNA uptake but also for the specific cleavage of viral genomes ...

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Section: Discussionsupporting

confidence: 66%

mentioning

confidence: 71%

Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

Albright

Nellissery

Szczepaniak

et al. 2011

J Virol

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“…That these polypeptides represent internal components might be inferred by their under-representation in light nucleocapsids and presence in heavy capsids, which include virus cores (Gibson & Roizman, 1972). One of them may be equivalent to protein 22a, a constituent of heavy nucleocapsids of both HSV-1 and HSV-2 (Gibson & Roizman, 1972Roizman, , 1974Heilman et al, 1979). However, this correlation has not been established experimentally.…”

Section: Discussionmentioning

confidence: 99%

“…However, we cannot rule out the possibility that NC2 is distributed more widely on the capsid but is more accessible to antibody, for topographical reasons, near the vertices. Because disulphide bonds, which appear to be necessary for the maintenance of capsid morphology (McCombs & Williams, 1973), are responsible for a structural affiliation between NC 1 and NC2 of HSV-2 capsids (Zweig et al, 1979), it is probable that NC2 is bound to NC 1 within the capsid structure.…”

Section: Discussionmentioning

confidence: 99%

Morphological Components of Herpesvirus. III. Localization of Herpes Simplex Virus Type 1 Nucleocapsid Polypeptides by Immune Electron Microscopy

Vernon¹,

Leon

Cohen

et al. 1981

Journal of General Virology

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S U M M A R YHerpes simplex virus type 1 (HSV-1) nucleocapsids were observed in the electron microscope after their reaction with IgG's purified from the sera of rabbits immunized with the individual nucleocapsid polypeptides. The combining sites of NC1, the major capsid protein (mol. wt. 154K), were distributed over the entire capsid surface. This result provides further evidence that NC 1 represents the major hexamer constituent. NC2 (mol. wt. 50K) was less widely distributed and appeared to be located at capsid vertices; that antigen may be a constituent of the pentamers or of peripentameric hexamers. One or both of NC3 and NC4 (mol. wt. 40K and 38K) were also located all over the capsid, possibly at positions interior to those of NC 1. One or both may represent the intercapsomeric fibrils, hexamer-associated protein or material associated with the pericore. The locations of the other nucleocapsid polypeptides could not be determined.

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“…Mr standards ranged from 205K (myosin) to 43K (ovalbumin) (Bio-Rad). Two-dimensional (diagonal) gel electrophoresis in the presence and absence of reducing agent in the first dimension was performed by the method of Zweig et al (1979), except that 9% DATD cross-linked gels were used in both dimensions. Tube gels which were not reduced during electrophoresis in the first dimension were soaked in 50 mM-Tris-HCl pH 6.8, 0.1% SDS and 10 m~I-DTT for 30 rain at room temperature, prior to electrophoresis in the second dimension.…”

Section: Methodsmentioning

confidence: 99%

Identification of the gB Homologues of Equine Herpesvirus Types 1 and 4 as Disulphide-linked Heterodimers and Their Characterization Using Monoclonal Antibodies

Meredith

Stocks

Whittaker

et al. 1989

Journal of General Virology

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SUMMARYEquine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of Mr 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of Mr 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent Mr of 108K (140K under nonreducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a l12K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

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Identification of disulfide-linked protein complexes in the nucleocapsids of herpes simplex virus type 2

Cited by 36 publications

References 11 publications

Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

Morphological Components of Herpesvirus. III. Localization of Herpes Simplex Virus Type 1 Nucleocapsid Polypeptides by Immune Electron Microscopy

Identification of the gB Homologues of Equine Herpesvirus Types 1 and 4 as Disulphide-linked Heterodimers and Their Characterization Using Monoclonal Antibodies

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