Comparative whole genome sequence analysis of wild-type and cidofovir-resistant monkeypoxvirus

Abstract

We performed whole genome sequencing of a cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl) cytosine] [HPMPC]}-resistant (CDV-R) strain of Monkeypoxvirus (MPV). Whole-genome comparison with the wild-type (WT) strain revealed 55 single-nucleotide polymorphisms (SNPs) and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV). These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV) species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies.

Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

“…In previous studies, the A314V change (Table 1) was found in VACV HPMPC R (i) as a single point mutation (10) or (ii) associated with other amino acid modifications, including H296Y, H319N, S338F, and R604S (43). In HPMPC R MPXV, the mutated residue A314V has been reported with another amino acid change at position 684 (A684T), and they were concomitant to A613T and T808M (30). The A684V change has already been selected with another amino acid change at position 314 (A314T), and these mutations were involved in (S)-HPMPC and (S)-HPMPDAP resistance development in VACV (Table 1) (4,32).…”

“…To measure the growth rates of recombinant viruses, HEL cells were allowed to grow until confluence in 24-well plates and infected with the strain of interest at an MOI of 0.01. At different time points, i.e., 3,6,9,24,30,48, and 72 h postinfection, the cells were harvested (in triplicates), and the viruses were released by freeze-thawing for further titrations by plaque assays in HEL cells. Virus titers, expressed in PFU/ml, are presented as the means from two independent experiments.…”

“…Other studies further described the genotyping of VACV resistant to (S)-HPMPC (HPMPC R ) (4,10,43) and to (S)-HPMPDAP (HPMPDAP R ) (32). Several amino acid changes, listed in Table 1, have been mapped to the E9L protein of drug-resistant VACV and MPXV, though the specific roles of some of these mutations have not been uniformly elucidated (4,10,30,32,43). Various degrees of resistance to (S)-HPMPC or (S)-HPMPDAP, ranging from 3-to 25-fold increase in EC 50 s, may be achieved depending on the nature and the number of the mutated allele(s) (4,10,32,43).…”

“…However, the recent identification of mutated residues in the DNA polymerase of MPXV HPMPC R at amino acid positions that are similar to those seen in VACV HPMPC R might predict for conserved genetic alterations within OPVs under (S)-HPMPC pressure (30). In order to extend our comprehension of how specific mutations in the DNA polymerase of different OPVs affect the susceptibility to ANPs, we studied the impact of homologous mutations within E9L of CMLV and VACV.…”

Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-Oriented Synthesis Library

“…Compounds that show promise in preclinical or clinical settings are the cidofovir derivative CMX001 (24), which is a nucleoside analog, and ST-246 (12,13), which has a unique mechanism of action in preventing viral egress from infected cells. With the majority of inhibitors, including CMX001 and ST-246, viral resistance is achieved rapidly in cell culture (1,9,33). As with many antimicrobial strategies, effective treatment is likely to require combination therapy.…”