Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis

Abstract

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNAbinding protein (65KDBP), the major DNA-binding protein (infected cel polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA-ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication proteins to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.

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

“…Localization of epitope-tagged UL5, UL8, and UL52 in infected cell nuclei. UL29, UL30, UL42, and UL9 all localize to replication compartments (14,30,31). Olivo et al were unable to detect a subnuclear localization of UL5, UL8, and UL52 with polyclonal antisera in an indirect immunofluorescence assay, perhaps because of the low sensitivity of the antisera they used (30).…”

“…A polymerase null mutant virus forms prereplicative sites in the absence of PAA. Prereplicative sites can be found in the nuclei of cells infected with temperature-sensitive UL30 or temperature-sensitive UL42 mutants at the nonpermissive temperature or with a null mutant in UL30 (2,14,31). Thus, inactivation of the polymerase complex with drugs or by means of mutation leads to prereplicative site formation.…”

“…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).…”

Two regions of the herpes simplex virus type 1 UL42 protein are required for its functional interaction with the viral DNA polymerase

“…ICP8 has also been reported to stimulate pol activity twofold (36,45), and one group has reported that it is required for UL42 to increase processivity of pol (26). Moreover, all three proteins colocalize to the same replication compartments in productively infected cells (2,11,22,23,43). It has been shown previously that the carboxy-terminal (C-terminal) 227 amino acids of pol are involved in and sufficient for complex formation with UL42 on the basis of coimmunoprecipitation of in vitro transcription-translation products with pol-specific antibody (13).…”

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…”