Dataset Information

Proline and tyrosine residues in scaffold proteins of herpes simplex virus 1 critical to the interaction with portal protein and its incorporation into capsids.

ABSTRACT: Previous results showed that amino acids 449 to 457 of pU(L)26, a component of the scaffold of herpes simplex virus 1 capsids, were critical for interaction with the portal protein encoded by U(L)6 and for incorporation of the portal into capsids. To identify residues in this scaffold domain critical for the interaction with pU(L)6, the two proteins were coexpressed in the absence of other viral proteins and subjected to immunoprecipitation with scaffold-specific antibody. Coimmunoprecipitation of pU(L)6 was precluded by pU(L)26 mutations Y451A, P452A, and E454A but not by P449A, R456A, or Y450A. In infected cells, Y451A and P452A diminished solubilization of pU(L)6, reduced incorporation of the portal into the capsid, and precluded viral replication and DNA packaging. In contrast, E454A did not affect these parameters despite the fact that E454 is invariant in a number of different alphaherpesvirus scaffold proteins. These data suggest that the interaction between the scaffold E454A mutant and portal protein is rescued by other viral functions. Finally, we show that amino acids 448 to 459 of pU(L)26 are sufficient to interact with pU(L)6 in a glutathione S-transferase pulldown assay in the absence of other viral proteins and that this interaction is inhibited with excess peptide containing pU(L)26 amino acids 443 to 462. Together, these observations suggest that a direct interaction between this scaffold domain and portal protein mediates incorporation of the portal into the capsid.

SUBMITTER: Yang K

PROVIDER: S-EPMC2715747 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Proline and tyrosine residues in scaffold proteins of herpes simplex virus 1 critical to the interaction with portal protein and its incorporation into capsids.

Yang Kui K Baines Joel D JD

Journal of virology 20090527 16

Previous results showed that amino acids 449 to 457 of pU(L)26, a component of the scaffold of herpes simplex virus 1 capsids, were critical for interaction with the portal protein encoded by U(L)6 and for incorporation of the portal into capsids. To identify residues in this scaffold domain critical for the interaction with pU(L)6, the two proteins were coexpressed in the absence of other viral proteins and subjected to immunoprecipitation with scaffold-specific antibody. Coimmunoprecipitation ...[more]

PMID: 19474093

Similar Datasets

Project description:Herpes simplex virus replicates in the nucleus, where new capsids are assembled. It produces procapsids devoid of nucleic acid but containing the preVP22a scaffold protein. These thermo-unstable particles then mature into A-, B- or C-nuclear icosahedral capsids, depending on their ability to shed the proteolytically processed scaffold and incorporation of the viral genome. To study how these viral capsids differ, we performed proteomics studies of highly enriched HSV-1 A-, B- and C-nuclear capsids, relying in part on a novel and powerful flow virometry approach to purify C-capsids. We found that the viral particles contained the expected capsid components and identified several tegument proteins in the C-capsid fraction (pUL21, pUL36, pUL46, pUL48, pUL49, pUL50, pUL51 and pUS10). Moreover, numerous ribosomal, hnRNPs and other host proteins, absent from the uninfected controls, were detected on the capsids with some of them seemingly specific to C-capsids (glycogen synthase, four different keratin-related proteins, fibronectin 1 and PCBP1). A subsequent proteomics analysis was performed to rule out the presence of protein complexes that may share similar density as the viral capsids but do not otherwise interact with them. Using pUL25 or VP5 mutant viruses incapable of assembling C-nuclear or all nuclear capsids, respectively, we confirmed the bulk of our initial findings. Naturally, it will next be important to address the functional relevance of these proteins.IMPORTANCE Much is known about the biology of herpesviruses. This includes their unique ability to traverse the two nuclear envelopes by sequential budding and fusion steps. For HSV-1, this implies the pUL31/pUL34 and pUL17/pUL25 complexes that may favor C-capsid egress. However, this selection process is not clear, nor are all the differences that distinguish A-, B- and C-capsids. The present study probes what proteins compose these capsids, including host proteins. This should open up new research avenues to clarify the biology of this most interesting family of viruses. It also reiterates the use of flow virometry as an innovative tool to purify viral particles.

Project description:The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit-length HSV genome. Here, biochemical and structural studies focused on the unique portal vertex of wild-type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal proteins pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

Dataset Information

Proline and tyrosine residues in scaffold proteins of herpes simplex virus 1 critical to the interaction with portal protein and its incorporation into capsids.

Publications

Proline and tyrosine residues in scaffold proteins of herpes simplex virus 1 critical to the interaction with portal protein and its incorporation into capsids.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets