Our data suggest that CPXV prevents antigen presentation to CD8+ T cells by expressing two unrelated and independently acting proteins. CPXV012 limits the TAP-dependent supply of peptides to the MHC-I peptide-loading complex. Since empty MHC-I molecules are retained in the ER by Tapasin (Ortmann et al., 1997
; Schoenhals et al., 1999
) the observed ER-retention by CPXV012 is thus an indirect consequence of inhibiting peptide loading. Also, the reduction of MHC-I steady state levels is likely the result of the ultimate degradation of empty MHC-I molecules as reported for cells expressing ICP47 (Hughes et al., 1997
). In contrast, CPXV203 inhibits the intracellular transport of MHC-I independent of its peptide loading status via direct binding and KDEL-mediated retrieval (Byun et al., 2007
). However, although each protein acts independently, CPXV expressing both ORFs was more efficient in preventing T cell stimulation than SKOs (). Moreover, both ORFs are co-expressed as immediate early genes (supplemental Fig. 2
). A possible scenario is that MHC-I escaping CPXV012 (either due to incomplete TAP inhibition, or TAP-independent peptide loading or as empty molecules) are then retrieved by CPXV203. This sequential interference could conceivably take place in a complex consisting of CPXV012, Tap and MHC-I that also binds CPXV203 thus optimizing prevention of antigen presentation.
Remarkably, CPXV012 and CPXV203 prevent ER-exit of both human and mouse MHC-I. This is in stark contrast to the limited species specificity observed for most other viral MHC-I inhibitors, particularly TAP-inhibitory proteins (Ahn et al., 1996
). In many instances, this restriction correlates with the limited host range of the corresponding viruses. In contrast, CPXV infects many mammalian species and this is reflected in its ability to inhibit both human and mouse MHC-I pathways. The availability of two independently acting MHC-I inhibitors might thus also increase the host range for CPXV. Furthermore, by independently targeting MHC-I and TAP, CPXV also counteracts the polymorphism of MHC-I. Proteins that interact directly with MHC-I often differentially affect polymorphic MHC-I alleles. Examples are Adenovirus E19 (Feuerbach et al., 1994
), HCMV US2 and US11 (Barel et al., 2006
; Machold et al., 1997
), and MCMV m06 and m152 (Wagner et al., 2002
). In contrast, TAP represents a conserved target.
So far, TAP-inhibitors were only described for the herpesvirus family. Although not every herpesvirus inhibits TAP, TAP-inhibitors were identified for members of each the α, β and γ subfamily. Interestingly, most herpesviral TAP-inhibitors are unrelated to each other suggesting that TAP inhibition has evolved independently multiple times. The repeated occurrence of TAP-inhibition within the herpesvirus family correlates with the ability of most herpesviruses to establish longterm persistent infections within their hosts. The acquisition of multiple TAP-inhibitors probably reflects the enormous immunological pressure exerted by CD8+ T cells during long-term infection by herpesviruses. In contrast, OPXVs are typically acute viruses that employ a “hit and run” strategy to disseminate within a host population. While OPXV-infection often causes significant morbidity, the host develops long-term protective immunity once the infection is cleared (Hammarlund et al., 2003
). This protective immunity is considered to be mostly antibody mediated (Edghill-Smith et al., 2005
). However, the fact that CPXV devotes two of its genes to counteract CD8+ T cells suggests that the cytolytic T cell immune response also plays a major role in controlling acute infection with CPXV.
Interestingly, CPXV012 of CPXV-BR seems to be derived by truncation and mutation from the D10L protein of other CPXV strains. D10L contains a C-type lectin domain homologous to host Clr-b, a ligand for the NK cell inhibitory receptor NKR-P1B (Voigt et al., 2007
). While it is not known whether D10L activates this inhibitory receptor, NK cell inhibition was demonstrated for the related protein RCTL in rat CMV (Voigt et al., 2007
). CPXV-BR thus seems to have modified an NK cell inhibitory protein to become a T cell evasion protein. This “exaptation” of unrelated viral proteins for TAP-inhibition is reminiscent of the UL49.5 protein which functions as a chaperone for the viral gM protein in all herpesviruses, but TAP-inhibition has been observed only for a few varicelloviruses (Koppers-Lalic et al., 2005
). That TAP can be inhibited by several different proteins of different evolutionary origin and in entirely unrelated viral families is probably facilitated by several possible modes of TAP-inhibition. As ABC transporter, TAP pumps peptides across the ER-membrane by ATP-hydrolysis. Several of the multiple steps that are involved in this process can be experimentally distinguished such as peptide binding, ATP binding and ATP-hydrolysis. So far, TAP inhibitors can be classified as inhibitors of peptide binding (ICP47 (Ahn et al., 1996
)), inhibitors of ATP-binding (US6, EHV UL49.5) (Hewitt et al., 2001
; Koppers-Lalic et al., 2005
) or both (BNLF2a) (Horst et al., 2009
). Additionally, TAP-degradation has been reported for BHV-1 U49.5 (Koppers-Lalic et al., 2005
). While further work will be required to determine the mechanism of TAP-inhibition by CPXV012 in more detail, its type II transmembrane topology together with the fact that the ER-luminal domain seems to be more sensitive to manipulation than the cytosolic domain renders it likely that CPXV012 will not prevent peptide-binding to TAP which occurs in the cytoplasm. In fact, both inhibitors of peptide binding, ICP47 and BNLF2a, represent short 60–70 amino-acid proteins that act on the cytosolic face of TAP. In contrast, EHV UL49.5 and HCMV US6 both inhibit ATP-binding, which is coupled to peptide translocation, and represent type I transmembrane proteins that act on the luminal side of TAP. It is thus possible that CPXV012 will similarly block ATP binding.
Truncated gene fragments similar to CPXV012 are widely found in poxviral genomes (www.poxvirus.org
) and frequently represent loss-of-function phenotypes. Our finding suggests that such truncated ORFs might acquire a entirely new, unanticipated functions. It is thus important to consider this evolutionary “genetic debris” when considering poxviral proteomes and their associated function. Since TAP homologues are present in all vertebrate genomes and since all vertebrates are potential hosts of herpes and poxviruses, our data further suggest that TAP-inhibition might be more wide-spread than previously thought, particularly in large DNA viruses.