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

Abstract

As previously shown, 11 loci are required to complement human cytomegalovirus (HCMV) DNA replication in a transient-transfection assay (G. S. Pari and D. G. Anders, J. Virol. 67:6979-6988, 1993). Six of these loci encode known or candidate replication fork proteins, as judged by sequence and biochemical similarities to herpes simplex virus homologs of known function; three encode known immediate early regulatory proteins (UL36-38, IRS1/TRS1, and the major immediate early region spanning UL122-123); and two encode early, nucleus-localized proteins of unknown functions (UL84 and UL112-113). We speculated that proteins of the latter five loci might cooperate to promote and regulate expression of the six replication fork proteins. To test this hypothesis we made luciferase reporter plasmids for each of the replication fork gene promoters and measured their activation by the candidate effectors, expressed under the control of their respective native promoters, using a transient-cooperativity assay in which the candidate effectors were subtracted individually from a transfection mixture containing all five loci. The combination of UL36-38, UL112-113, IRS1, or TRS1 and the major immediate early region produced as much as 100-fold-higher expression than the major immediate early region alone; omitting any one of these four loci from complementing mixtures produced a significant reduction in expression. In contrast, omitting UL84 had insignificant (less than twofold), promoterdependent effects on reporter activity, and these data do not implicate UL84 in regulating HCMV early-gene expression. Most of the effector interactions showed significant positive cooperativity, producing synergistic enhancement of expression. Similar responses to these effectors were observed for the each of the promoters controlling expression of replication fork proteins. However, subtracting UL112-113 had little if any effect on expression by the UL112-113 promoter or by the simian virus 40 promoter-enhancer under the same conditions. Several lines of evidence argue that the cooperative interactions observed in our transient-transfection assays are important to viral replication in permissive cells. Therefore, the data suggest a model in which coordinate expression of multiple essential replication proteins during permissive infection is vitally dependent upon the cooperative regulatory interactions of proteins encoded by multiple loci and thus have broad implications for our understanding of HCMV biology.

Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays

“…or create a local environment suitable for efficient synthesis of viral DNA replication proteins in the naturally permissive HF cells. Note that they also failed to associate any transactivation function with UL84 expression, consistent with our interpretation that its role in replication is potentially direct (42). Thus, the requirement for such transactivators in DNA replication can most likely be explained by a dependence on cooperative regulatory interactions of multiple loci for the efficient expression of cellular and viral replication proteins.…”

“…Additionally, the removal of UL112-113 from the replication assay severely reduced the detectable level of oriLyt amplification. Recently, Iskenderian et al (42) argued that the cooperative interactions observed between some of the auxiliary factors are critical to either produce a synergistic enhancement of HCMV early promoters 7408 SARISKY AND HAYWARD J. VIROL. or create a local environment suitable for efficient synthesis of viral DNA replication proteins in the naturally permissive HF cells.…”

Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins

“…The HCMV UL84 is a nonstructural early protein which has been shown to interact with the HCMV immediate-early protein IE86 (23,66). UL84 is also required in the complementation assay for replication of a plasmid containing the HCMV origin of replication (25,48). Interestingly, the 6.9-kb transcript, which likely encodes the M84 product, is detectable only at 8 h p.i., consistent with early gene expression.…”

Access of viral proteins to mitochondria via mitochondria‐associated membranes

“…The HCMV UL36-38 locus is required for its oriLyt DNA replication [101][102][103][104]. Expression of nuclear genes, including induction of the human heat shock protein 70 (hsp70) and selected HCMV early genes, can be regulated by pUL37x1\vMIA and gpUL37 [78,105,106].…”