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PMID: 9060678 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays.

Journal of virology ·Vol. 71 ·No. 4 ·1997-04-00 ·Pages 3146-60

Zhong L, Hayward GS

Abstract

Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.

MeSH Terms
Animals Bromodeoxyuridine Chlorocebus aethiops DNA Helicases/genetics,metabolism DNA Primase DNA Replication DNA, Viral/biosynthesis DNA-Binding Proteins/genetics,metabolism DNA-Directed DNA Polymerase Exodeoxyribonucleases Gene Expression Regulation, Viral Genes, pol Herpesvirus 1, Human/drug effects,genetics,physiology Humans Immediate-Early Proteins/genetics Phosphonoacetic Acid/pharmacology Plasmids Replication Origin Transfection Tumor Suppressor Protein p53/metabolism Vero Cells Viral Proteins/genetics,metabolism Virus Assembly
Chemicals
DNA, Viral DNA-Binding Proteins ICP27 protein, human herpesvirus 1 ICP8 protein, Simplexvirus Immediate-Early Proteins Tumor Suppressor Protein p53 Viral Proteins herpes simplex virus, type 1 protein ICP4 UL9 protein, Human herpesvirus 1 DNA Primase DNA-Directed DNA Polymerase Exodeoxyribonucleases helicase-primase, Human herpesvirus 1 DNA polymerase, Simplexvirus DNA Helicases Bromodeoxyuridine Phosphonoacetic Acid
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zhong L
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Hayward G S
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.71.4.3146-3160.1997
Published
1997-04-00
Pages
3146-60
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC191447
Subset
IM
Grants
NCI NIH HHS · R01 CA28473 · United States
NCI NIH HHS · R01-CA34 · United States
NIGMS NIH HHS · T32GM07445 · United States
External Links
PubMed source DOI: 10.1128/JVI.71.4.3146-3160.1997 PMC Full Text View journal details
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