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PMID: 8386266 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Journal of virology ·Vol. 67 ·No. 5 ·1993-05-00 ·Pages 2575-82

Pari GS, Kacica MA, Anders DG

Abstract

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.

MeSH Terms
Cells, Cultured Cosmids/genetics Cytomegalovirus/genetics,growth & development DNA Replication DNA, Viral/biosynthesis Genetic Complementation Test Humans Immediate-Early Proteins/genetics,metabolism Membrane Glycoproteins Open Reading Frames Trans-Activators Transcriptional Activation Viral Envelope Proteins Viral Proteins/genetics,metabolism Virus Replication
Chemicals
DNA, Viral IE1 protein, cytomegalovirus IE2 protein, Cytomegalovirus Immediate-Early Proteins Membrane Glycoproteins TRS1 protein, Human herpesvirus 5 Trans-Activators UL115 protein, Human herpesvirus 5 UL36 protein, Human herpesvirus 1 UL36-38 protein, Cytomegalovirus Viral Envelope Proteins Viral Proteins glycoprotein H, Cytomegalovirus glycoprotein H, Human cytomegalovirus glycoprotein O, cytomegalovirus
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Pari G S
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Kacica M A
Anders D G
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.67.5.2575-2582.1993
Published
1993-05-00
Pages
2575-82
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC237578
Subset
IM
Grants
NIAID NIH HHS · AI31249 · United States
NIAID NIH HHS · AI33416 · United States
External Links
PubMed source DOI: 10.1128/JVI.67.5.2575-2582.1993 PMC Full Text View journal details
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