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

Poxviruses are large, enveloped, pleomorphic dsDNA viruses that infect a diverse array of mammals, reptiles, and insects [1]. The causative agent of Smallpox, Variola virus (VARV) is a member of the OPV genus. Smallpox was declared eradicated in 1980, however, natural or illicit re-emergence poses a risk for a growing non-vaccinated population [2]. MPV is a re-emerging pathogen within the OPV genus that causes sporadic outbreaks in monkeys and humans in West and Central Africa and, recently, in North America [3]. MPV can cause human disease clinically similar to Smallpox but with lower morbidity and mortality rates [4]. Although terrestrial and arboreal rodents and mammals are thought to play a role in MPV transmission, human to human transmission is known to occur [5].

Poxviruses possess large, complex genomes that encode their own viral replication machinery in addition to a plethora of immunomodulating proteins [1]. The major components of the poxviral replication complex include the poxvirus DNA polymerase (DNApol, E9L), transcription factor heterodimer (vETF), DNA-dependent RNA polymerase, RNA polymerase accessory protein (RAP94), viral poly-A polymerase (VP55/VP39), capping methyltransferase (D1/D10), and the DNA polymerase processivity factor (A20) [1, 6]. Chemotherapeutic strategies for poxvirus infection have largely targeted viral DNA synthesis in order to disrupt the virus replication cycle [7, 8].

A number of nucleoside/nucleotide analogs are available that inhibit OPVs [7]. The acyclic nucleoside phosphonate analogue (S)-1-[3-hydroxy-2-phosphonyl-methoxypropyl)] cytosine ((S)-HPMPC) or cidofovir (CDV) has been shown to inhibit in vitro viral replication of most known DNA viruses including poxviruses [9‐11]. Recent studies suggest a mechanism whereby CDV may allosterically reposition the 3' nucleophile of terminal and short +strand synthesis products leading to aberrant chain extension [12, 13]. Using the VACV DNApol E9L, previous studies indicate CDV incorporation slows chain elongation and inhibits DNA synthesis [12]. In addition, CDV has been shown to inhibit 3' to 5' exonuclease activity of E9L when incorporated in the penultimate position relative to the primer terminus [12]. By altering chain extension CDV affects DNA synthesis, a key regulator of poxvirus gene expression. Thus, alterations in gene expression and replication are likely to occur during CDV exposure, and, could result in mutations affecting conserved determinants of the virus life cycle.

Cidofovir activity appears to be conserved in dsDNA viruses providing a common strategy for inhibiting viral replication in important human diseases caused by these virus families [14, 8, 15]. Substitutions in the DNApol exonuclease (A314T) and polymerase (A684V) domains of the VACV DNA polymerase have previously been mapped and shown to confer CDV resistance [16, 17]. CDV resistant strains in other members of the OPV genus, including MPV, Camelpoxvirus (CMPV), and Cowpoxvirus (CWPV) have already been reported [15]. DNApol mutations conferring resistance to CDV may be conserved among non-VACV OPV species although, presently, such sequence analyses have not been performed. Indeed, a portion of resistance attributes are likely to be conserved across dsDNA viruses. A number of additional features of CDV-resistance remain uncharacterized. CDV resistant strains frequently display an attenuated phenotype [18, 15] through yet uncharacterized natural genetic alterations. In addition, it has been suggested that, in some cases, resistance to CDV requires mutations outside the DNA polymerase. One previous study identified a CDV-R VACV which exhibited a single non-essential substitution in the DNApol that upon reconstruction did not confer CDV resistance [18]. To date, such loci elsewhere in the genome remain unknown. Whole-genome sequence data could provide valuable insight into breadth of mutations induced by CDV exposure and yield insight into further requisites for attenuation and resistance.

We report here the first whole genome sequence of a CDV-R poxvirus. Our data revealed a plethora of substitutions within the CDV-R MPV genome, one-third of which were distributed throughout the viral replication machinery. Substitutions identified in the MPV DNA polymerase are consistent with those previously observed in VACV suggesting CDV-resistance determinants may be conserved in the OPV genus. The numerous substitutions observed throughout the replication and RNA processing machinery suggest multiple accrued mutations may alter the timing and regulation of the virus life cycle under CDV exposure. Novel loci reported here may inform future studies aimed at mechanistic interaction of CDV with the replication complex.

Whole genome comparison of CDV-R and WT strains of Monkeypox revealed 55 single nucleotide polymorphisms (SNPs) including four insertions, six deletions, and 44 nucleic acid substitutions (Table 1, Figure 1, 2). A total of 10 intergenic and 45 intragenic SNPs, were observed that include 17 synonomous, 26 nonsynonomous substitutions and one tandem repeat contraction (Table 1). Over a third of all observed SNPs occurred within genes involved in virus replication and DNA metabolism. The physical distribution of all observed SNPs and indels (insertions/deletions) are illustrated in Figure 1.

In the current study we report the complete genomic sequence of a CDV-R strain of MPV. In addition, we present a focused and comparative bioinformatic analysis that revealed predicted alterations in topological features of functionally active domains within essential virus proteins. Previous data indicate mutations at sites 314 and 684 in the DNApol represent the primary determinants of CDV-R in VACV [15, 20]. Although second-site substitutions elsewhere in the VACV genome have been implicated previously in a CDV-R clone [18], they have yet to be identified. The present study may provide clues to the location of such mutations. The MPV DNApol mutations reported here provide the first indication that CDV-R loci previously identified in VACV are perhaps conserved in fully-virulent, non vaccine strains, though such speculation must await experimental validation. Such data may inform efforts in development of Smallpox-related medical countermeasures. Any direct effects of selected mutations reported here on the resistant or attenuated phenotype of MPV must await future determination. These regions may be of particular interest for future site-directed mutagenesis studies to dissect 1) potential yet-uncharacterized mutations elsewhere in the genome that may play a role in the CDV-R phenotype, and, 2) the genetic basis of the characteristic attenuated phenotype of CDV-R poxviruses. It is possible the substitutions observed in our analysis outside the viral DNA polymerase, for example in the RNA polymerase and mRNA capping enzyme, may contribute to the resistant or attenuated phenotype of CDV-R MPV. Such changes may represent compensatory, adaptive, or attenuating variations in gene expression or replication. Also, adaptive substitutions which support a CDV-R phenotype may result in alterations in the timing of the viral gene expression program that could reduce fitness compared to wild-type yet sustain gene expression in the presence of CDV. Both adaptive and non-adaptive substitutions may also be facilitated through mutator alleles in the DNA or RNA polymerases. As DNA synthesis is a key regulator of gene expression in poxviruses, it is possible the aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and replication that must be overcome by a resistant strain. The genome sequence of CDV-R MPV may inform future research into the mechanism of action of CDV as well as dissection of the phenotypic properties of resistant poxviruses. Furthermore, defining the potential contribution of substitutions in the replication complex and RNA processing machinery may inform current therapeutic development strategies and yield further insight into CDV-resistance and attenuation.