Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8

Abstract

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous end-joining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.

FIG. 1.

Coprecipitation of cellular and viral proteins with ICP8 using the anti-ICP8 39S monoclonal antibody. (A) IPs of ICP8-associated proteins from mock-infected (lane 1) or wt HSV-1-infected (lane 2) HEp-2 cells were resolved in an SDS-8% polyacrylamide gel and then stained with Coomassie blue. The proteins indicated at the right were identified by MS. The molecular mass markers are shown at the left in kilodaltons. (B) Immunoprecipitations were performed in the presence or absence of EtBr and then an equal volume of each lysate or IP was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blot was probed with antibodies against the indicated cellular or viral proteins. Lane 1, mock (M) lysate; Lane 2, wt (WT) KOS lysate; Lane 3, wt KOS lysate with EtBr; Lane 4, mock IP; lane 5, wt KOS IP; lane 6, wt KOS IP with EtBr; lane 7, mock lysate; lane 8, wt KOS lysate; lane 9, mock IP; lane 10, wt KOS IP; lane 11, mock IP with EtBr; lane 12, wt KOS IP with EtBr.

FIG. 2.

Distribution of cellular proteins in HSV-1-infected cells. HEp-2 cells infected with wt KOS or mock infected were processed for immunofluorescence at 6.5 h postinfection. The first panel (left) of each protein series shows the protein distribution in mock-infected cells. The next panel (middle) shows the distribution of the cellular protein in an HSV-1-infected cell. The last panel (right) shows the distribution of ICP8 in the same cell as that in the middle panel.