Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.     

Identification and characterization of equine herpesvirus type 1 pUL56 and its role in virus-induced downregulation of major histocompatibility complex class I

Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein which is expressed as different forms and predominantly localizes to Golgi membranes. In addition, the transmembrane (TM) domain of the type II membrane protein was shown to be indispensable for correct subcellular localization and a proper function. pUL56 by itself is not functional with respect to interference with MHC-I and likely needs another unidentified viral protein(s) to perform this action. Surprisingly, pUL49.5, an inhibitor of the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses, appeared not to be required for pUL56-induced early MHC-I downmodulation in infected cells. In conclusion, our data identify a new immunomodulatory protein, pUL56, involved in MHC-I downregulation which is unable to perform its function outside the context of viral infection.     

The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.     

Downregulation of class I major histocompatibility complex surface expression by varicella-zoster virus involves open reading frame 66 protein kinase-dependent and -independent mechanisms

We show here that the varicella-zoster virus (VZV) open reading frame 66 (ORF66) protein kinase is one mechanism employed to reduce class I major histocompatibility complex (MHC-I) surface expression in VZV-infected cells. Cells expressing enhanced green fluorescent protein-tagged functional and inactivated ORF66 (GFP-66 and GFP-66kd) from replication-defective adenovirus vectors revealed that ORF66 reduced MHC-I surface levels in a manner dependent on kinase activity. Cells infected with recombinant VZV expressing GFP-66 exhibited a significantly greater reduction in MHC-I surface expression than that observed in cells infected with VZV disrupted in GFP-66 expression. MHC-I maturation was delayed in its transport from the endoplasmic reticulum through the Golgi in both adenovirus-transduced cells expressing only GFP-66 and in VZV-infected cells expressing high levels of GFP-66, and this was predominantly kinase dependent. MHC-I levels were reduced in VZV-infected cells, and analyses of intracellular MHC-I revealed accumulation of folded MHC-I in the Golgi region, irrespective of ORF66 expression. Thus, the ORF66 kinase is important for VZV-mediated MHC-I downregulation, but additional mechanisms also may be involved. Analyses of the VZV ORF9a protein, the ortholog of the bovine herpesvirus 1 transporter associated with antigen processing inhibitor UL49.5 revealed no effects on MHC-I. These results establish a new role for viral protein kinases in immune evasion and suggest that VZV utilizes unique mechanisms to inhibit antigen presentation.     

The gene 10 (UL49.5) product of equine herpesvirus 1 is necessary and sufficient for functional processing of glycoprotein M

The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-beta-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyA Delta 49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyA Delta 49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyA Delta 49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M(r) protein was coprecipitated with gM in KyA- or KyA Delta 49.5R-infected cells or virions. This protein was absent in cells infected with Ky Delta 49.5 or KyA Delta gM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.     

The varicellovirus-encoded TAP inhibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1(b) revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.     

Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH₂-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37_NH2 and gpUL37_COOH were both detected in the ER and MAM fraction, even though only pUL37_NH2 is preferentially imported into mitochondria but gpUL37_COOH is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH₂-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid

Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.     

Signaling of a varicelloviral factor across the endoplasmic reticulum membrane induces destruction of the peptide-loading complex and immune evasion

Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.     

The human cytomegalovirus protein UL37 exon 1 associates with internal lipid rafts

The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-β-cyclodextrin (MβCD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria.     

HIV-1 Nef disrupts antigen presentation early in the secretory pathway

Human immunodeficiency virus, type 1 Nef disrupts viral antigen presentation and promotes viral immune evasion from cytotoxic T lymphocytes. There is evidence that Nef acts early in the secretory pathway to redirect major histocompatibility complex class I (MHC-I) from the trans-Golgi network to the endolysosomal pathway. However, a competing model suggests that Nef acts much later by accelerating MHC-I turnover at the cell surface. Here we demonstrate that Nef targets early forms of MHC-I molecules in the endoplasmic reticulum by preferentially binding hypophosphorylated cytoplasmic tails. The Nef-MHC-I complex migrates normally into the Golgi apparatus but subsequently fails to arrive at the cell surface and become phosphorylated. Cell type-specific differences in the rate of MHC-I transport through the secretory pathway correlate with responsiveness to Nef and co-precipitation of adaptor protein 1 with the Nef.MHC-I complex. We propose that the assembly of a Nef.MHC-I.adaptor protein 1 complex early in the secretory pathway is important for Nef activity.     

Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

Herpes simplex virus type 1 glycoprotein K and the UL20 protein are interdependent for intracellular trafficking and trans-Golgi network localization

Final envelopment of the cytoplasmic herpes simplex virus type 1 (HSV-1) nucleocapsid is thought to occur by budding into trans-Golgi network (TGN)-derived membranes. The highly membrane-associated proteins UL20p and glycoprotein K (gK) are required for cytoplasmic envelopment at the TGN and virion transport from the TGN to extracellular spaces. Furthermore, the UL20 protein is required for intracellular transport and cell surface expression of gK. Independently expressed gK or UL20p via transient expression in Vero cells failed to be transported from the endoplasmic reticulum (ER). Similarly, infection of Vero cells with either gK-null or UL20-null viruses resulted in ER entrapment of UL20p or gK, respectively. In HSV-1 wild-type virus infections and to a lesser extent in transient gK and UL20p coexpression experiments, both gK and UL20p localized to the Golgi apparatus. In wild-type, but not UL20-null, viral infections, gK was readily detected on cell surfaces. In contrast, transiently coexpressed gK and UL20p predominantly localized to the TGN and were not readily detected on cell surfaces. However, TGN-localized gK and UL20p originated from endocytosed gK and UL20p expressed at cell surfaces. Retention of UL20p to the ER through the addition of an ER retention motif forced total ER retention of gK, indicating that transport of gK is absolutely dependent on UL20p transport. In all experiments, gK and UL20p colocalized at intracellular sites, including the ER, Golgi, and TGN. These results are consistent with the hypothesis that gK and UL20p directly interact and that this interaction facilitates their TGN localization, an important prerequisite for cytoplasmic virion envelopment and egress.     

Bovine herpesvirus 1 UL49.5 protein inhibits the transporter associated with antigen processing despite complex formation with glycoprotein M

Bovine herpesvirus 1 (BHV-1) interferes with peptide translocation by the transporter associated with antigen processing (TAP). Recently, the UL49.5 gene product of BHV-1 was identified as the protein responsible for the observed inhibition of TAP. In BHV-1-infected cells and virions, the UL49.5 protein forms a complex with glycoprotein M (gM). Hence, it was investigated whether UL49.5 can combine the interactions with gM and the TAP complex. In cell lines constitutively expressing both UL49.5 and gM, UL49.5 appears to be required for functional processing of gM. Immunofluorescence-confocal laser scanning microscopy demonstrated that both proteins are interdependent for their redistribution from the endoplasmic reticulum to the trans-Golgi network. Remarkably, expression of cloned gM results in the abrogation of the UL49.5-mediated inhibition of TAP and prevents the degradation of the transporter. However, in BHV-1-infected cells, differences in UL49.5 and gM expression kinetics were seen to create a window of opportunity at the early stages of infection, during which time the UL49.5 protein can act on TAP without gM interference. Moreover, in later periods, non-gM-associated UL49.5 can be detected in addition to the UL49.5/gM complex. Thus, it has been deduced that different functions of UL49.5, editing of gM processing and inhibition of TAP, can be combined during BHV-1 infection.     

An intrinsic network of polar interactions is responsible for binding of UL49.5 C-degron by the CRL2KLHDC3 ubiquitin ligase

Bovine herpesvirus type 1 (BoHV-1) is a pathogen of cattle responsible for infectious bovine rhinotracheitis. The BoHV-1 UL49.5 is a transmembrane protein that binds to the transporter associated with antigen processing (TAP) and downregulates cell surface expression of the antigenic peptide complexes with the major histocompatibility complex class I (MHC-I). KLHDC3 is a kelch domain-containing protein 3 and a substrate receptor of a cullin2-RING (CRL2) E3 ubiquitin ligase. Recently, it has been identified that CRL2^KLHDC3 is responsible for UL49.5-triggered TAP degradation via a C-degron pathway and the presence of the degron sequence does not lead to the degradation of UL49.5 itself. The molecular modeling of KLHDC3 in complexes with four UL49.5 C-terminal decapeptides (one native protein and three mutants) revealed their activity to be closely correlated with the conformation which they adopt in KLHDC3 binding cleft. To analyze the interaction between UL49.5 and KLHDC3 in detail, in this work a total of 3.6 μs long molecular dynamics simulations have been performed. The complete UL49.5-KLHDC3 complexes were embedded into the fully hydrated all-atom lipid membrane model with explicit water molecules. The network of polar interactions has been proposed to be responsible for the recognition and binding of the degron in KLHDC3. The interaction network within the binding pocket appeared to be very similar between two CRL2 substrate receptors: KLHDC3 and KLHDC2.     

The varicellovirus UL49.5 protein blocks the transporter associated with antigen processing (TAP) by inhibiting essential conformational transitions in the 6+6 transmembrane TAP core complex

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.     

The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.