The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase

Abstract

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.

Fig. 1.

Synthesis of HCMV DNA in the presence of AIC246 or ganciclovir. (A) HCMV AD169-infected cells were treated with placebo, AIC246, or ganciclovir (GCV). Total intracellular DNA was harvested during a period of 96 h postinfection as indicated, and viral progeny DNA was measured by quantitative real-time PCR. All experiments were performed in triplicate, and standard deviations are depicted by error bars. (B) Production of progeny virus at the indicated time points monitored via virus yield measurements.

Fig. 2.

Effect of AIC246 on expression of representative immediate-early (IE), early (E), and late (L) proteins of HCMV. (A) Immunofluorescence analyses. HCMV AD169-infected fibroblast cells were treated with either GCV (d to f) or AIC246 (g to i) or were kept untreated (a to c). Cells were fixed at the indicated time postinfection (pi) and stained for IE1 (a, d, and g), UL44 (b, e, and h), or MCP (c, f, and i) expression (red signals). Cell nuclei were counterstained using DAPI (blue signals). (B) Western blot analyses. NHDF cell monolayers were infected with HCMV strain AD169 at an MOI of 0.1 PFU/cell. The infection was allowed to proceed in the absence (−) or presence (+) of AIC246. At different time points pi (h/pi), cells were harvested and processed for Western blotting. Representative HCMV IE, E, or L proteins indicated at the right of each panel were visualized using the respective antibodies. Cellular β-actin served as a loading control (lc). Size markers (in kDa) are indicated on the left. M, mock-infected cells.

Fig. 3.

Markerless introduction of point mutations into ORF UL56 via BAC mutagenesis (marker transfer). (A) Schematic representation of the UL56 domain organization according to Champier et al. (11). Conserved regions are indicated as light gray boxes (I to XII) and variable regions (VR1 and VR2) as dark gray boxes. The localizations of the independently inserted UL56 mutation t723c (L241P) and g1107c (R369S) are highlighted by asterisks. (B) EcoRI or NcoI restriction analysis of WT BAC pHG (lanes 1 and 6) and recombinant BACs pHG-UL56-L241P (lanes 2, 4, 7, and 9) and pHG-UL56-R369S (lanes 3, 5, 8, and 10), respectively. DNA was size fractionated by a 0.9% agarose gel. Asterisks indicate new EcoRI or NcoI restriction fragments resulting as intermediates from recombination. (C) Single-step growth curve of reconstituted viruses RV-HG (WT), RV-HG-UL56-R369S (UL56-R369S), and RV-HG-UL56-L241P (UL56-L241P). NHDF cultures were infected at an MOI of 0.1 PFU/cell. At the indicated time points postinfection, supernatants were harvested and titrated via IE1 staining. Standard deviations are derived from three independent experiments.

Fig. 4.

Functional viral DNA cleavage assay. (A) Schematic model of HCMV DNA synthesis and packaging. The HCMV terminase complex (UL56/UL89) and the HCMV genome structure are illustrated. Long and short arms are comprised of unique long (UL) and unique short (US) regions separated by a region designated the L/S junction. pac indicates the terminase cleavage site at the genome terminus. Below is an expansion of the location of the KpnI restriction sites at genomic transitions and the terminase cleavage site. A gray rectangle indicates the position of the terminal DNA probe. Following restriction digestion, concatemeric viral DNA yields an ∼8.4-kb KpnI fragment, whereas terminase-cleaved concatemeric DNA results in a shortened ∼4-kb fragment. (B) Southern blot analysis of viral DNA. HELF cells were infected with HCMV AD169 or AD169-rAIC246-1 and maintained at the AIC246 concentrations indicated. BAY 38-4766- and BDCRB-treated cells served as positive controls. Seventy-two h after infection, viral DNA was isolated, digested with KpnI, and size fractionated by gel electrophoresis. After blotting, hybridization was carried out using the terminal DNA probe depicted in panel A. Terminase-cleaved and uncleaved genomic HCMV DNA can be distinguished by the size of the respective restriction fragment.

Fig. 5.

Transmission electron micrographs of thin-section preparations of virus-infected placebo- or AIC246-treated cells. Placebo (A and C)- or AIC246 (B and D)-treated, AD169-infected HFF cells were fixed 5 days postinfection, and thin sections were prepared for electron microscopic analysis. Panels A and B show a representative image of a cell nucleus, and panels C and D show an image of the respective cytoplasm. Arrows in the nucleus indicate A, B, and C capsids, respectively; arrows in the cytoplasm indicate virions.