Analysis of Human Cytomegalovirus ori Lyt Sequence Requirements in the Context of the Viral Genome

Abstract

During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.Human cytomegalovirus (HCMV), like all herpesviruses, has at least two different modes of infection, either lytic replication or latency. Whereas the replication mode of the HCMV genome during latency is rather elusive, substantial progress has been made in dissecting the molecular steps that occur during lytic DNA replication (35, 39; for a review, see reference 41). Following cell entry, HCMV capsids are transported to the nuclear pores, where they release the linear genomes into the cell nucleus. Then, the linear genomes are rapidly circularized and serve as templates for transcription and replication. It has been proposed that the initial steps in DNA replication of herpesviruses take place by a theta-like mechanism, followed by a rolling-circle mechanism of replication at later stages of the lytic infection cycle (reviewed in references 36 and 46). This view of herpesvirus DNA replication has recently been challenged by the observation that genome circularization seems not to be a prerequisite for replication of herpes simplex virus (HSV) and the suggestion that linear genomes serve as replication templates (31). It remains to be shown whether the same applies to HCMV and other herpesviruses as well.Regardless of which mechanism is used, lytic replication of herpesviruses initiates at defined regions of the viral genomes, th...

The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

“…The recombinant viruses (RV) used in this study were derived from the bacterial artificial chromosome (BAC)-cloned genome (pHB5) of the HCMV laboratory strain AD169 (6). The HCMV BAC pHG contains an enhanced green fluorescent protein (EGFP) gene in the unique short region and lacks the open reading frames (ORFs) UL1 to UL10 (UL1-10), with only one Flp recognition target (FRT) site retained at this position (9). The BAC pHD corresponds to pHB5 with the UL1-10 locus deleted.…”

“…Primers used to disrupt the UL52 gene in the HCMV BACs were UL52-ko.for (5Ј-GCGGCCGCCTCATACCAGGTAA ATCCTCAACACCCCGCCAAGAAAAGTGCCACCTGCAGAT-3Ј) and UL52-ko.rev (5Ј-TTGGTGACGCGGATGTTGCCGGCGCACTGCGGGTCG CAGAACAGGAACACTTAACGGCTGA-3Ј), and plasmid pOri6K-F5 served as a template. pOri6K-F5 encodes a knR marker flanked by mutant FRT sites that do not interact with the wild-type FRT sites (9). Subsequent excision of the FRT-flanked KnR marker was done in E. coli via Flp recombinase expressed by plasmid pCP20 as described previously (5).…”

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

“…Having shown that pUL77-mGFP and pUL93 are capsid constituents, we next asked whether these proteins may play a role in HCMV genome encapsidation. To this end, either UL77 or UL93 was deleted from the HG genome, an AD169 strain-based EGFP-expressing HCMV BAC (31). We first tested if genome cleavage is still carried out after disruption of UL77 or UL93.…”

“…pHB5 denotes the original AD169 BAC, and pHG represents an enhanced green fluorescent protein (EGFP)-expressing pHB5 derivative (31). hTERT-RPE-1 cells (Clontech) were propagated as described previously (29).…”

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

“…Thus, it is speculated that HCMV establishes an environment in which it hijacks the cellular DNA replication machinery to benefit its own genome replication. HCMV DNA replication initiates from the replication origin oriLyt [24][25][26] and is thought to take place at sites near the nuclear domain 10 (ND10), which is disrupted as the replication progresses [27,28]. Subsequently, HCMV replication sites are enlarged to form globular replication compartments that eventually fill the nucleus at late stage [19,29,30].…”