This study identifies the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel class of viral immune evasion proteins. The UL49.5 gene products interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides in the ER lumen through inhibition of TAP. Within the UL49.5 family of TAP inhibitors, heterogeneity is observed with respect to the mechanisms that underlie TAP inhibition. Whereas BHV-1 UL49.5 targets TAP for proteasomal degradation 
, PRV and EHV-1 UL49.5 do not diminish the steady state levels of TAP1 or TAP2. Interestingly, EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP, a function that is not influenced by BHV-1 or PRV UL49.5. All TAP-inhibiting UL49.5 proteins arrest the transporter complex in a translocation-incompetent state.
UL49.5 homologs are encoded by all Herpesviridae
analyzed to date 
. However, the TAP-inhibiting capacities of these proteins appear to be restricted to certain members of the genus Varicellovirus
. Members of this virus genus have co-evolved with their respective host species 
. Viruses of even-toed ungulates or Artiodactyla
like BHV-1 and PRV co-evolved with cattle and pigs; viruses of odd-toed ungulates or Perissodactyla
(EHV-1 and EHV-4) with horses; the carnivore viruses FHV-1 and CHV with cats and dogs, and the Old World primate virus VZV with humans 
(). The identification of the UL49.5 proteins encoded by BHV-1, PRV, EHV-1, and EHV-4 as members of the UL49.5 family of TAP inhibitors suggests that more UL49.5 proteins with this property may be found in varicelloviruses of even- and odd-toed ungulate hosts. Considering the shared evolution of (herpesviruses from) carnivores and (herpesviruses from) odd-toed ungulates 
, CHV UL49.5 was expected to inhibit TAP as effectively as EHV-1 and EHV-4 UL49.5. However, the reduction of TAP-dependent peptide transport caused by CHV UL49.5 was very moderate compared to the inhibition by the other TAP-inhibiting UL49.5 proteins. The identification of the UL49.5 domains contributing to TAP inhibition will provide more insights into these differences.
Phylogenetic tree of alphaherpesvirus UL49.5 proteins constructed on the basis of all presently known UL49.5 amino acid sequences of Alphaherpesvirinae using ClustalV.
VZV infection of human cells results in reduced expression of MHC class I at the cells surface 
. The VZV ORF66-encoded serine-threonine protein kinase has been shown to be one of the VZV proteins contributing to MHC class I down-regulation in VZV-infected cells 
. However, a VZV recombinant lacking a functional ORF66 product still causes down-regulation of MHC class I surface expression, indicating that additional modulators of MHC class I-restricted antigen presentation are encoded by VZV. The observed down-regulation of MHC class I surface expression on VZV-infected cells 
is not induced by UL49.5 when expressed individually. Despite the observed interaction between VZV UL49.5 and the peptide-loading complex, this protein alone did not block peptide transport by TAP and it had no effect on antigen recognition by HLA-A1 and HLA-A2-restricted CTL clones. As VZV occupies a somewhat isolated position in the phylogenetic tree of varicelloviruses (), it seems likely that evolutionary divergence has influenced VZV to acquire a separate mechanism to interfere with MHC class I-restricted antigen presentation. Alternatively, UL49.5 might co-operate with another unidentified VZV-encoded protein in order to reduce antigen presentation by MHC class I molecules. During virus infection, UL49.5 can be found in a complex with glycoprotein M (gM). However, the co-expression of VZV UL49.5 and glycoprotein M has no effect on the expression of MHC class I molecules at the cell surface 
, indicating that gM does not act as a modulator of UL49.5 with respect to TAP inhibition.
Interaction with the conserved viral membrane glycoprotein M appears to be a common property of all UL49.5 homologs, as is the presence of a single cysteine residue in their ER-luminal/extracellular domain 
. This cysteine residue is involved in the interaction of UL49.5 with gM, with which it forms a disulfide-linked heterodimers 
. The complex of UL49.5 and gM is implicated in virion maturation and membrane fusion processes 
. Interestingly, the interaction of BHV-1 UL49.5 with gM interferes with its capacity to block TAP 
. Nevertheless, UL49.5 blocks peptide transport by TAP in BHV-1-infected cells. This may be explained by the fact that UL49.5 is expressed prior to and in excess of the early-late gM 
Interference with TAP-mediated peptide transport is an effective way of reducing CTL recognition and is used by several other herpesviruses, including HSV-1 and -2, HCMV, MHV-68, and EBV 
. Compared to the other herpesvirus-encoded TAP inhibitors, the cross-species activity of UL49.5 proteins is remarkable. Except for CHV UL49.5, the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 all exhibit the ability to target human TAP. In addition, BHV-1 UL49.5 inhibits peptide transport by murine 
, rat, equine, and porcine TAP (D.K.L. and M.V., unpublished observations). Human, porcine, bovine, and rodent TAP1 and TAP2 demonstrate a substantial degree of amino acid identity (70–80%) 
. Thus, the ability of UL49.5 proteins to act across species barriers most likely relies on structural homology within the TAP domains critically involved in UL49.5-TAP interaction. Apparently, this is less so for the domains within TAP that are targeted by US6, mK3 and BNLF2a, whose actions seem to be restricted largely to the natural host species. BHV-1 UL49.5 reduces TAP protein levels in bovine, human, and murine cells, and also mediates degradation of human TAP in insect cells when co-expressed with UL49.5 
, indicating conservation of the pathway involved in this degradation process.
The UL49.5 proteins exhibit unexpected differences in their mechanisms of TAP inhibition, despite their close evolutionary relatedness. The cytoplasmic domain of BHV-1 UL49.5 is essential for mediating degradation of both human and bovine TAP. EHV-1 and PRV UL49.5 have no influence on the stability of TAP. Apparently, degradation of TAP is facilitated by a yet unknown signal within the C-terminal domain of BHV-1 UL49.5, which is not present in the other homologs. Studies to identify the nature of this sequence motif are in progress.
The interaction of EHV-1 and EHV-4 UL49.5 with TAP blocks ATP binding to TAP. This feature distinguishes EHV UL49.5 from the other homologs studied. Interestingly, removal of the cytoplasmic domain of the EHV-1 UL49.5 protein did not restore the ability of TAP to bind ATP. Therefore, a direct interaction of EHV-1 UL49.5 with the cytosolic nucleotide binding domains of TAP is unlikely. Instead, the viral protein appears to arrest TAP in a translocation-incompetent state, incompatible with ATP-binding. This may resemble the type of structural change caused by HCMV US6 
. US6, a type I transmembrane protein, interacts with the luminal side of the TAP transporter and blocks ATP-binding by prohibiting essential conformational rearrangements within TAP. The inability of the BHV-1 and PRV UL49.5 homologs to interfere with ATP-binding could be due to a slightly different conformational change induced by these proteins.
Based on the results presented in this study, the UL49.5 proteins encoded by BHV-1, PRV, EHV-1, and EHV-4 can be classified as a new family of TAP-inhibiting proteins. These proteins share the ability of inducing a conformational arrest of TAP, which results in impaired peptide transport and inhibition of MHC class I-restricted antigen presentation. In view of these joint features it is likely that the TAP inhibiting UL49.5 proteins originate from a common ancestral protein, which acquired this capacity earlier during evolution. The VZV UL49.5 protein may be a rudimentary form with respect to TAP inhibition, or it may have lost its TAP inhibitory capacity later on. Alternatively, it may require additional VZV proteins for the inhibition of TAP.
This study has revealed unexpected variation among UL49.5 proteins of varicelloviruses with respect to their mechanisms of TAP inhibition. These differences can be related to distinct evolutionary pathways of these varicelloviruses. The UL49.5 family of TAP-inhibiting proteins does not demonstrate any structural or functional similarity to TAP-inhibiting proteins encoded by other herpesviruses, for instance ICP47, US6, mK3, or BNLF2a. This diversity of TAP-inhibiting proteins acquired by distantly related members of the subgroups of alpha-, beta-, and gammaherpesviruses is remarkable and presents a striking example of functional convergent evolution. At the same time, this identifies TAP as an Achilles' heel of the MHC class I antigen presentation pathway. Inhibition of TAP has apparently provided a strong advantage to these herpesviruses during co-evolution with their hosts.