Human cytomegalovirus terminase inhibitors letermovir, tomeglovir, and the halogenated benzimidazoles modify DNA cleavage/packaging to generate a super-genomic DNA species by cleavage site skipping.

Human cytomegalovirus (HCMV) genome packaging involves terminase-mediated translocation of concatemeric viral DNA into procapsids and cleavage to release C-capsids containing mature viral genomes. Three classes of terminase inhibitors are active against HCMV, represented by BDCRB, tomeglovir, and letermovir. Approval of letermovir for prevention of HCMV disease in stem cell transplant patients validated terminase as an important antiviral target. Like tomeglovir and BDCRB, we show that letermovir induces formation of a monomer-plus (M+) supergenomic viral DNA. In contrast to the HCMV genome, in which L and S segments are separated by an internal cleavage site, we propose that M+ has an S-L-S structure that results from skipping of the terminal cleavage site, packaging of an additional S segment, and cleavage at the next cleavage site encountered in the concatemer. In support of this model an HCMV lacking an internal cleavage site failed to form M+ and the ratio of sequencing reads from S and L segments in M+ was elevated ∼ two-fold compared to normal genomic DNA. C-capsids were observed within infected-cells treated with letermovir or BDCRB, albeit at three-fold lower prevalence. However, all viral DNA species formed in the presence of letermovir or BDCRB (including M+) were sensitive to nuclease digestion. These findings suggest that, despite structural differences, terminase inhibition involves altered cleavage site recognition, resulting in packaging of abnormal DNA species within nuclease-permeable capsids. Discovery of additional terminase inhibitors may reveal distinct mechanisms of inhibition and perhaps novel terminase functions.