Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Abstract

Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter

“…after 6 days post-infection (52). The IE gene products, IE1 and IE2, have been shown to be required for initiating viral replication (28,29,53,54). The results from our study and the previous recombinant CMV assays (51,52) suggest the possible involvement of PDX1 in human CMV replication through the transcriptional regulation of IE gene expression.…”

Estrogen-related receptor α is required for efficient human cytomegalovirus replication

“…6) and protein levels (Fig. 5) were reduced at 48 and 72 hpi, and this could in turn reduce the levels of other viral gene products, because IE2 is a potent transcriptional activator that drives the cascade of HCMV gene expression (54). The reduced level of IE2 following knockdown or pharmacological inhibition of ERRα argues that the nuclear receptor is required for its optimal expression during the late phase of the replication cycle.…”

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

“…Viral proteins involved in HCMV DNA replication at least include UL44 (the DNA polymerase processivity factor), UL84 (likely a replication initiator interacting with the RNA/DNA hybrid within oriLyt) and the immediate-early 2 (IE2) protein [29,[31][32][33][34]. HCMV with IE2 gene deleted is not viable [35]. IE2 is a well studied transactivator essential for the induction of a cascade of HCMV lytic genes [36], some of which directly participate in viral DNA replication.…”