Myxoma virus M156 is a specific inhibitor of rabbit PKR but contains a loss-of-function mutation in Australian virus isolates

Abstract

Myxoma virus (MYXV) is a rabbit-specific poxvirus, which is highly virulent in European rabbits. The attenuation of MYXV and the increased resistance of rabbits following the release of MYXV in Australia is one of the best-documented examples of host-pathogen coevolution. To elucidate the molecular mechanisms that contribute to the restriction of MYXV infection to rabbits and MYXV attenuation in the field, we have studied the interaction of the MYXV protein M156 with the host antiviral protein kinase R (PKR). In yeast and cell-culture transfection assays, M156 only inhibited rabbit PKR but not PKR from other tested mammalian species. Infection assays with human HeLa PKR knock-down cells, which were stably transfected with human or rabbit PKR, revealed that only human but not rabbit PKR was able to restrict MYXV infection, whereas both PKRs were able to restrict replication of a vaccinia virus (VACV) strain that lacks the PKR inhibitors E3 and K3. Inactivation of M156R led to MYXV virus attenuation in rabbit cells, which was rescued by the ectopic expression of VACV E3 and K3. We further show that a mutation in the M156 encoding gene that was identified in more than 50% of MYXV field isolates from Australia resulted in an M156 variant that lost its ability to inhibit rabbit PKR and led to virus attenuation. The species-specific inhibition of rabbit PKR by M156 and the M156 loss-of-function in Australian MYXV field isolates might thus contribute to the species specificity of MYXV and to the attenuation in the field, respectively.oxviruses are large double-stranded DNA viruses that exclusively replicate in the cytoplasm of infected cells. Members of the Poxviridae family can productively infect a wide variety of animal hosts. Interestingly, the binding and entry of poxviruses into cells is largely independent of host species after which virus replication is initiated. The successful completion of virus replication, however, depends on the effective subversion of the host cell's innate immune responses (1). Even closely related poxviruses can exhibit drastic differences in their host ranges. Whereas some poxviruses have only one host species, such as variola virus, the causative agent of smallpox, which is restricted to humans, others, such as cowpox and monkeypox viruses, can infect many different species and thus display very broad host ranges (2). A number of poxvirus genes have been discovered that influence the host range and cell tropism of poxviruses and have therefore been termed "host range genes" (3, 4). Although the molecular mechanisms responsible for their host range functions have not been elucidated in detail, it is clear that most poxviral host range proteins interact with components of the host immune system and that host species-specific interactions likely play a major role.Myxoma virus (MYXV) is a poxvirus that belongs to the genus leporipoxvirus and shows a restricted host range infecting only leporids (rabbits and hares). MYXV is highly lethal to European (E.) rabbits causing case fatality rate...

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia

“…However, the transcription start sites of most MYXV mRNAs have not been mapped and hence actual expression may differ from that assigned [39, 31, 40]. In the UK sequences, mutations upstream of the M000 .…”

Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype

“…The latter two proteins are important virulence factors (39,40). In addition, along with other viruses, BD44 possesses a mutation L71P in M156 (an inhibitor of the antiviral protein kinase R), which has been demonstrated to cause loss of function (41). Thus, the loss of major virulence functions is compatible with high virulence, suggesting compensatory mutations are playing an important role.…”

Species‐specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus

“…(knockdown resistant) (human), pSG5-mmPKR (mouse), and pSG5-oaPKR (sheep) were previously described. 9,19 Bos taurus (cow) PKR was cloned from MDBK cells and the sequence was identical to NP_835210.2. Capra hircus (goat) PKR (XM_005686488.2) was synthesized (GENEWIZ) and subcloned from the pUC57 vector into pSG5 to generate pSG5-chPKR.…”

“…Similarly, we showed that inactivation of M156 inhibited virus replication in rabbit cells. 19 However, very little is known about these PKR-inhibitor interactions in other types of poxviruses.…”