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

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.

Section: Resultssupporting

confidence: 78%

Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

“…6F), suggesting that maturation may be alleviating the negative impact of foscarnet on LC proliferation and/or viability. The specific bases of these toxic effects are difficult to pinpoint, as foscarnet was reported to only minimally affect the activity of host DNA polymerases at concentrations that completely abrogate viral DNA replication (63). Indeed, no cell loss was observed in treated HFF cultures when viral progeny production was completely blunted (Fig.…”

Section: Cd34mentioning

confidence: 98%

Dynamics of Human Cytomegalovirus Infection in CD34⁺Hematopoietic Cells and Derived Langerhans-Type Dendritic Cells

Coronel¹,

Takayama²,

Juwono³

et al. 2015

Acquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV but can easily be targeted by orally administered vaccines, while their bone marrow resident progenitors are important sites of virus latency. In this work, we tracked the progress of infection in CD34 ؉ progenitor cells, immature LC (iLC), and mature LC (mLC) exposed to the clinicallike strain TB40-BAC4 or to the vaccine strain AD169varATCC, prior to their long-term maintenance under either immature or mature conditions. We show that the genomes of both strains are efficiently maintained in CD34؉ cells during their differentiation into iLC, although this requires the presence of larger amounts of input AD169varATCC DNA. Lipopolysaccharide-and CD40 ligand-induced maturation of iLC derived from latently infected progenitors was not associated with robust viral genome replication and progeny production, while maturation of directly infected iLC increased and prolonged expression of the viral immediate early proteins. While effective replication of viral genomes from both strains occurred only in mLC, both iLC and mLC produced viral progeny, suggesting that both types of LC may contribute to CMV horizontal transmission in vivo. H uman cytomegalovirus (CMV), a major cause of disease and death in immunocompromised and in congenitally infected individuals, is normally acquired by contact between infected bodily fluids such as urine and saliva and host mucosal surfaces, particularly those lining the oral and nasal cavities. IMPORTANCE Human CMV is usually acquired via the oral and nasal mucosae. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of these tissues. Understanding how CMV interacts with LC and their hematopoietic progenitors is thus essential to develop innovative means of defense against this virus. Here we show that the genomes of a viru-From these peripheral sites, CMV is thought to reach the circulation and hence the bone marrow, where latency is established in myeloid cells, including CD34 ϩ hematopoietic progenitors (1-5). Reactivation from latency in the myeloid progeny of these cells is then believed to be the source of newly produced viral particles, which upon amplification in oral epithelial cells are released in the saliva and contribute to CMV horizontal transmission in vivo (6-8).Immature Langerhans-type dendritic cells (iLC) are the only professional antigen-presenting cells located in the outermost layers of the oral mucosa (9-14). As such, iLC are bound to be the first immune cells to encounter invading pathogens that access their hosts via the oral cavity, such as CMV. Upon contact with "danger" signals, iLC migrate toward the draining lymph nodes and begin the process of maturation, which culmina...

“…The pulse times began at 5 s and increased to 60 s with a 3:1 forward-tobackward ratio. DNA species were visualized by ethidium bromide staining and UV light and then transferred to Nytran nylon membranes (Schleicher & Schuell) and hybridized to specifically detect MCMV DNA using 32 P-labeled DNA from plasmid pON4048 as previously described (19).…”

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

confidence: 99%

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

Wang¹,

Nixon

McVoy³

2008

Self Cite

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...