Virions of members within the family Poxviridae are large and complex, contain a double-stranded DNA genome of 130–375 kbp, and virus replication takes place in the cytosol [1]. Among poxviruses, especially the genus Orthopoxvirus (OPV) contains several important pathogens, and endemic cowpox viruses (CPXV) are the most common cause of zoonotic OPV infections in Europe and parts of northern and central Asia today [2, 3].
Understanding CPXV pathogenesis has shifted focus on the vast array of poxvirus-encoded accessory proteins which give each poxvirus its unique characteristics of immunomodulation and pathogenesis [4,5,6,7]. Among these, the so-called host range genes are supposed to be responsible for differences in tropism and host range between individual poxviruses [6,7,8].
The p28 protein of poxviruses belongs to the KilA-N/RING domain-containing p28/N1R protein family [6,7,8]. The p28 protein was first described as a virulence factor of ectromelia virus (ECTV) [9]. Disruption of the p28 gene abolished lethality of ECTV for susceptible mice, although it had no effect on virus replication in several other cell types, with the exception of mouse peritoneal macrophages [9, 10]. The ECTV p28 gene was found to be highly conserved, and orthologues of p28 are found in the genomes of most OPV, with the notable exception of several strains of vaccinia virus (VACV) [6, 9, 11]. Furthermore, orthologues of the p28 gene are also present in several other genera of the Chordopoxvirinae [6, 12].
The full-length p28 protein consists of 242 amino acids and combines an N-terminal KilA-N domain, which acts as DNA-binding domain [13], and a C-terminal RING domain [6]. The protein is translated in the early phase of viral replication, is present throughout the viral life cycle and localizes to cytoplasmic virus factories facilitated by its KilA-N domain [9, 10, 14]. The mechanism of how p28 contributes to viral host range is currently not understood. In vitro studies showed that the p28 proteins of VACV strain IHD-W, ECTV strain Moscow, variola virus strain Bangladesh-1975 and myxoma virus strain Lausanne possess E3 ubiquitin ligase activity which was attributed to the RING domain [15, 16]. Mutation of the RING domain stabilizes p28, indicating that p28 regulates itself via autoubiquitination and subsequent proteasomal degradation [14, 16]. Furthermore, p28 seems to be ubiquitinated by a yet unknown cellular ubiquitin ligase [14]. As the RING domain of p28 has been shown to be essential for the functionality of p28 as a virulence and host range factor of ECTV [9, 10], the mechanism of how p28 contributes to viral host range most likely depends on its function as a ubiquitin ligase. However, so far no cellular or viral substrates for p28-mediated ubiquitination despite p28 itself have been identified. Furthermore, the presence of genes encoding only KilA-N domains in other poxviruses makes it possible that this domain exerts an ubiquitin ligase-independent function that might contribute to host range and virulence [6].
Functionally, it has been speculated that p28 directs the ubiquitination and degradation of substrate host proteins which block viral replication through the induction of apoptosis [11, 16], as inhibition of apoptosis by p28 has been shown [17, 18]. Furthermore, as infection of murine macrophages with a p28 knockout ECTV resulted in a block of viral DNA replication and abortive infection following early viral gene expression [10], it was suggested that p28 might functionally compensate for an unknown cellular factor essential for viral DNA replication in macrophages.
In this study, we aimed to elucidate the significance of p28/N1R as a host range factor in CPXV. With regards to the importance of p28 for ECTV virulence, analysing the role of p28 in CPXV infection is of high interest concerning the understanding of CPXV pathogenesis. Therefore, we created recombinant CPXV which were either deficient in p28 expression or encoded mutant p28 proteins lacking a functional C-terminal RING domain. We show that CPXV lacking a functional p28 replicated less efficiently in macrophages of human or mouse origin, indicating that CPXV—like ECTV—is dependent on p28 to productively infect cells of the macrophage lineage. Given the importance of macrophages in containing systemic poxvirus dissemination [19], p28 may be important for CPXV virulence in vivo.
