Human Cytomegalovirus Origin-Dependent DNA Synthesis

Ellsmore, Victoria (2000) Human Cytomegalovirus Origin-Dependent DNA Synthesis. PhD thesis, University of Glasgow.

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Human cytomegalovirus (HCMV) origin-dependent DNA synthesis has not been as well studied as herpes simplex virus type 1 (HSV-1) DNA synthesis, partly due to the difficulty of manipulating HCMV permissive cell lines in tissue culture. Seven virus encoded proteins are required for HSV-1 origin-dependent DNA synthesis. Of the seven essential proteins, the six replication fork proteins, comprising the helicase/primase (UL5/UL8/UL52), the DNA polymerase (UL30/UL42) and a single stranded DNA binding protein (UL29), are conserved throughout the Herpesviridae. The seventh protein required for HSV-1 origin-dependent DNA synthesis is the origin-binding protein, UL9, which is conserved among the alphaherpesviruses and the roseolovirus genus. HCMV origin-dependent DNA synthesis requires the six conserved replication fork proteins and five other HCMV encoded proteins. HCMV does not encode an obvious counterpart to UL9. In this thesis, transient replication assays were used to further investigate the initiation of HCMV origin-dependent DNA synthesis. In order to simplify the transient transfection assay, alternatives to human fibroblast cell lines were tested for their ability to support HCMV DNA synthesis. A transfected HCMV origin-containing plasmid was replicated by HCMV strain AD 169 infection in Vero cells which did not apparently support the replication of the infecting genomes. The replication of independent origin-containing plasmids, and the failure of control plasmids to be replicated, confirmed that replication was specific to the HCMV origin sequences and independent of the vector used. Replication of an unmethylated origin-containing plasmid confirmed that the bacterial methylation patterns on the plasmid DNA were not responsible for its aberrant replication. Inhibitors were used to demonstrate that DNA synthesis was directed by the viral DNA polymerase. An assay exploiting the ability of simian virus 40 (SV40) T-antigen to recruit cellular DNA polymerases to direct SV40 origin-dependent DNA synthesis was used to confirm that the concentrations of inhibitors used did not abrogate the activity of the cellular (nuclear) replicative polymerases. In contrast to previously published data, genomic DNA synthesis was observed in HCMV strain ADI69 infected 293 cells and in HCMV strain Towne infected 293 and Vero cells. Therefore, the ability of HCMV to replicate in 293 and Vero cells was examined by virus growth assay and electron microscopic examination of infected cells. Neither Vero cells infected with HCMV strain AD 169 or Towne, nor 293 cells infected with HCMV strain AD 169, supported the production of HCMV progeny as measured by plaque formation or by electron microscopy (E.M.). HCMV particles were observed by E.M. at 140 h p.i. in 10% of 293 cells infected with HCMV strain Towne. An HCMV origin-containing plasmid was replicated by the HSV-1 replication fork proteins in the presence of the HCMV auxiliary proteins (TRS1, UL36-38, UL84, UL1 12-113 and UL122-123). Replication was dependent on intact origin sequences and was directed by the HSV-1 DNA polymerase. The HCMV major immediate early proteins (IE 1/2) were sufficient to mediate replication of an HCMV origin-containing plasmid by the HSV-1 replication fork proteins, addition of UL36-38 significantly increased the efficiency of replication. Stop mutants in pSVH (expressing the major immediate early proteins) and pZPS (expressing UL36-38) and a deletion mutant in pZP8 were used to demonstrate that IE2-86 and UL38 likely provided functions important for origin-dependent DNA synthesis. UL84, which specifies a putative origin-specific function (Sarisky & Hayward, 1996), was not required for HCMV origin-containing plasmid replication in this assay. Simian cytomegalovirus (SCMV) strain Colburn is more closely related to HCMV than HCMV is to HSV-1, therefore, the ability of SCMV superinfection to replicate an HCMV origin-containing plasmid was examined. SCMV infection directed the replication of an HCMV origin-containing plasmid in the absence of any HCMV proteins, addition of plasmids expressing UL84 did not increase the replication efficiency. The model system of replication of a varicella-zoster virus (VZV) origin-containing plasmid by HSV-1 superinfection was used to demonstrate that the addition of a cognate origin-binding protein to such "cross-complementation" assays increased replication efficiency. In an experiment in which the level replication of an HCMV origin-containing plasmid by SCMV strain Colburn was below the limits of detection, addition of HCMV IE1/IE2 and UL36-38 increased the efficiency of replication to a detectable level. The results of experiments using HSV-1 plasmids or SCMV virus to provide replication fork proteins to replicate an HCMV origin containing plasmid indicate that proteins provided by the IE1/IE2 locus and the UL36-38 locus, but not UL84, perform important origin specific roles in these assays.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: D McGeoch
Keywords: Virology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-76149
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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