Dramatic Effects of 2-Bromo-5,6-Dichloro-1-β-
            <scp>d</scp>
            -Ribofuranosyl Benzimidazole Riboside on the Genome Structure, Packaging, and Egress of Guinea Pig Cytomegalovirus

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Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-␤-D-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer؉ DNA that is unique to human cytomegalovirus. Analysis of monomer؉ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.The Herpesviridae family of viruses include several significant human pathogens, including herpes simplex virus type 1 (HSV-1) and -2, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and human cytomegalovirus (HCMV). These viruses have large (130-to 235-kb) double-stranded linear DNA genomes that circularize shortly after infection (22,38,39,49). Viral DNA is replicated to form large concatemers of head-to-tail-linked genomes. In a process termed genome maturation, the concatemeric DNA is packaged into capsids and cleaved to produce encapsidated unit length genomes (6,8,27,36,38,49,52,66).Genome maturation is highly conserved among the herpesviruses, but little is known about the mechanisms or the machinery involved. Striking similarities to the DNA packaging mechanisms of certain large double-stranded DNA bacteriophages exist, in particular, , T3, T4, and T7 (14). In both bacteriophages and herpesviruses, procapsids self-assemble around a protein scaffold. A unique vertex of the procapsid contains portal proteins arra...

supporting

confidence: 72%

Impact of 2-Bromo-5,6-Dichloro-1-β- d -Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation

McVoy

Nixon

2005

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“…8). Lower-molecular-weight smears of viral DNA were detected in samples from all four viruses and appeared similar to those observed previously for human cytomegalovirus (HCMV) (18) and guinea pig cytomegalovirus (24). Within these smears, no distinct 191-or 45-kb species could be distinguished, nor were differences evident between viruses containing functional or nonfunctional cleavage sites.…”

Section: Vol 82 2008 Cis-acting Sequences In Herpesvirus Genome Matsupporting

confidence: 54%

“…However, other studies suggest that pac2 or adjacent sequences may direct the initiation of DNA packaging, perhaps by binding proteins that mediate docking to procapsid portals, and that pac1 and pac2 independently govern formation of their respective termini (13,22,26,30). In addition, terminal sequences have been implicated as having a role in the duplication of terminal repeats (21) and in postpackaging capsid maturation events, such as the sealing or stabilization of DNAcontaining capsids or their targeting for nuclear egress (13,24). Indeed, using an HSV-1 amplicon system, Hodge et al found that mutation of pac1 sequences did not prevent packaging of DNA into intracellular capsids but rather gave rise to packaged amplicon genomes that appeared normal at their Uc (pac2) ends but lacked Ub (pac1) ends.…”

Section: Discussionmentioning

confidence: 99%

Definition of the Minimal cis -Acting Sequences Necessary for Genome Maturation of the Herpesvirus Murine Cytomegalovirus

Wang¹,

Nixon

McVoy³

2008

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Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large (ϳ180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of transacting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.Herpesviruses have large (130-to 235-kb) linear doublestranded DNA genomes that replicate via concatemeric intermediates consisting of head-to-tail-linked genomes (1,2,14,17,18,25,27,32). The concatemers are packaged into preformed capsids and cleaved at precise locations to release unit-length genomes within the capsids (3). The cis-acting sequences that are recognized by the cleavage/packaging machinery have not been fully defined, but two motifs denoted pac1 and pac2 were initially identified by their strong conservation near the ends of herpesvirus genomes (7). Typically, pac1 motifs consist of 3-to 7-bp A-or T-rich regions flanked on each side by short poly(C) tracts (7, 29), whereas pac2 motifs consist of 5-to 10-bp A-rich regions (7). In some viruses a consensus CGCGGCG motif is located just distal of pac2 (relative to the terminus) (7).In order to mutagenically define the cis cleavage/packaging sequences of a herpesvirus, we developed an assay based on cleavage at a second or "ectopic" cleavage site that was inserted into the murine cytomegalovirus (MCMV) genome. In 1984, Marks and Spector (15) showed that the two ends of the MCMV genome (designated X and C, respectively) ( Fig. 1) fuse shortly after infection to form "junctions." Upon concatemer synthesis, such junctions presumably serve as natural cleavage/packaging sites. We predicted tha...

“…This observation is not consistent with the assumption that AIC246 treatment results in concatemers remaining uncut and not being encapsidated. Recent findings of McVoy and Nixon may offer an explanation for this phenomenon (36,39). While analyzing the effect of BDCRB on HCMV genome maturation, the authors confirmed that BDCRB predominantly acts via blocking the processing of viral concatemer DNA to monomeric genome lengths (36,48).…”

Section: Discussionmentioning

confidence: 74%

The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase

Goldner

Hewlett

Ettischer

et al. 2011

296

241

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/ packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.