Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression

Abstract

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.

A Guide to Viral Inclusions, Membrane Rearrangements, Factories, and Viroplasm Produced During Virus Replication

“…Surrounded by vimentin, rough ER, mitochondria and polysomes. Chinchar et al, 1984;Darlington et al, 1966;Huang et al, 2006;Goorha, 1983, 1989;Zhao et al, 2007 Herpesviridae Barnard et al ., 1997;de Bruyn Kops et al ., 1998;Everett and Maul, 1994;Goodrich et al ., 1990;Jahedi et al ., 1999;Knipe et al, 1987;Lamberti and Weller, 1998;Leopardi et al ., 1997;Liptak et al, 1996;Markovitz and Roizman, 2000;Olivo et al ., 1989;Randall and Dinwoodie, 1986;Reynolds et al ., 2000;Taus et al ., 1998;Ward et al ., 1996 Contents of cellular origin RNA polymerase II, EAP ribosome component, proliferating cell antigen, retinoblastoma protein, p53, DNA ligase 1, DNA polymerase a, promyelocytic leukemia (PML), DNA-PKcs, Ku86 nonhomologous end joining, Bloom syndrome gene product, breast cancer-associated gene 1 protein, MSH2, Rad50, WRN RecQ helicase family member, BRG1 or BRM-associated factor 155, brahma-related gene-1 protein, brahma protein, histone deacetylase 2, hSNF2H, mSin3a, TATA binding protein (TBP), TBP-associated factors. Leopardi et al, 1997;Quadt et al, 2006;Taylor and Knipe, 2004;Wilcock and Lane, 1991 Nuclear sites of capsid assembly or assemblons…”

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication

“…A subset of the essential viral replication proteins have been shown to localize to replication compartments, specifically the origin-binding and polymerase complex proteins (14,30). In contrast, UL5, UL8, and UL52 were reported to be present in a diffuse staining pattern in infected nuclei (30). However, as noted by the authors, the polyclonal antisera used may not have been sensitive enough to detect the subnuclear localization of these nonabundant proteins.…”

“…HSV-1 DNA replication occurs in nuclear domains termed ''replication compartments,'' initially identified on the basis of UL29 staining patterns in an immunofluorescence assay (31); these compartments may represent the viral equivalent of cellular replicons. A subset of the essential viral replication proteins have been shown to localize to replication compartments, specifically the origin-binding and polymerase complex proteins (14,30). In contrast, UL5, UL8, and UL52 were reported to be present in a diffuse staining pattern in infected nuclei (30).…”

Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes

“…The finding that five additional loci were needed to complement HCMV DNA replication was surprising because homologous proteins were not required to complement HSV-1 or Epstein-Barr virus DNA synthesis (16,17,43). Moreover, none of these five showed evidence of similarity to the seventh essential HSV-1 replication component, the origin-binding protein UL9 (8,13,14).…”