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The signal sequences of the human cytomegalovirus (CMV) UL40 protein and its rhesus CMV (RhCMV) counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/V]L, VL9) that is presented by major histocompatibility complex (MHC) antigen E (MHC-E). The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts antigen processing and presentation and MHC Ia expression. This allows infected cells to maintain MHC-E surface expression and escape killing by Natural Killer cells. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation between the two proteins. Processing of both VL9 peptides is dependent on cleavage of their signal sequences by the host protease signal peptide peptidase. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 requires only its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the transporter associated with antigen processing. Our results also suggest that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by decreasing surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of Rh67, UL40 can partially counteract the up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, and further work will be required to determine if any such differences have implications for translating a RhCMV-vectored simian immunodeficiency virus (SIV) vaccine to HIV-1 using human CMV as a vector., Importance: The protective immune response induced by a rhesus cytomegalovirus (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine in rhesus macaques depends on the presence of the viral Rh67 gene in the vaccine. The Rh67 protein contains a peptide that allows the RhCMV-infected cells to maintain expression of major histocompatibility complex (MHC) antigen E at the cell surface. We show that production of this peptide, referred to as "VL9," mirrors that of the equivalent peptide present in the human cytomegalovirus (CMV) protein UL40, despite the little sequence similarity between the two CMV proteins. We also show that the mature UL40 and Rh67 proteins, which have no previously described function, also contribute to CMV immune evasion by reducing cell surface expression of MHC proteins important for the immune system to detect infected cells. Despite these similarities, our work also reveals possible differences between Rh67 and UL40, and these may have implications for the use of human CMV as the vector for a potential HIV-1 vaccine., Competing Interests: Oregon Health & Science University (OHSU) and K.F. have a substantial financial interest in Vir Biotechnology Inc., a company that may have a commercial interest in the results of this research and technology. K.F. is a co-inventor of OHSU patents licensed to Vir and received compensation for consulting for Vir. The potential individual and institutional conflicts of interest have been reviewed and managed by OHSU.

Vectors based on cytomegalovirus (CMV) represent a novel vaccine platform that maintains high frequencies of non-exhausted effector memory T cells in both CMV sero-positive and sero-negative individuals. In non-human primate models, CMV vectored vaccines provide unprecedented protection against simian immunodeficiency virus (SIV). Moreover, CMV vectors can be genetically altered to program highly diverse CD8+ T cell responses that differ in their epitope targeting including conventional, MHC-I restricted CD8+ T cells as well as unconventional CD8+ T cells restricted by MHC class II or non-polymorphic MHC-E. By modifying cytomegaloviral determinants that control unconventional T cell priming it is possible to uniquely tailor the CD8+ T cell response for each individual disease target in order to maximize prophylactic or therapeutic protection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Unlabelled: Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-κB. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-κB activation. We demonstrate that both viruses prevent ubiquitination of the NF-κB inhibitor IκBα, whereas SVV additionally prevents IκBα phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IκBα ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with β-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IκBα. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated β-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-κB pathway, since SVV with ORF61 deleted still prevented IκBα phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-α)-induced NF-κB activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection., Importance: The role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since infection of rhesus macaques by simian varicella virus (SVV) is used as an animal model of VZV infection, we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NF-κB, a central factor in eliciting proinflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments for VZV, since overcoming these mechanisms, either by small-molecule inhibition or by genetic modification of vaccine strains, is expected to reduce the pathogenic potential of VZV. Moreover, using SVV infection of rhesus macaques, it will be possible to study how increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.

Cytomegalovirus (CMV) efficiently evades many host immune defenses and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I (MHC-I) molecules in order to evade recognition and killing of infected cells by cytotoxic CD8(+) T cells. We recently showed that rhesus CMV-specific Rh178 intercepts MHC-I protein translation before interference of MHC-I maturation by homologues of the human CMV US6 family. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum, displaying a short luminal and large cytosolic domain, and that the membrane-proximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. Moreover, the transfer of amino-terminal fragments containing the MHC-I signal peptide renders recipient proteins susceptible to targeting by Rh178. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particle-dependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to SEC61-dependent protein translocation, since inhibition of MHC-I translocation by eeyarestatin 1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation.

Cytomegalovirus (CMV) can superinfect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. We have demonstrated that superinfection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally encoded inhibitors of major histocompatibility complex class I (MHC-I) antigen presentation, particularly the homologs of human CMV US2, 3, 6, and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo, thus supporting viral replication and dissemination during superinfection, a process that complicates the development of preventive CMV vaccines but that can be exploited for CMV-based vector development.

K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.

While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX(3)C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Galpha12 and Fractalkine through Galphaq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type-specific has important implications in the role of US28 in HCMV pathogenesis.

The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC) expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

Cullin RING ubiquitin ligases (CRULs) are found in all eukaryotes and play an essential role in targeting proteins for ubiquitin-mediated destruction, thus regulating a plethora of cellular processes. Viruses manipulate CRULs by redirecting this destruction machinery to eliminate unwanted host cell proteins, thus allowing viruses to slip past host immune barriers. Depending on the host organism, virus-modified CRULs can perform an amazing range of tasks, including the elimination of crucial signal transduction molecules in the human interferon pathway and suppression of virus-induced gene silencing in plants. This Perspective summarizes recent advances in our understanding of how viral proteins manipulate the function of CRULs.

Human cytomegalovirus (HCMV) is a paradigm for mechanisms subverting antigen presentation by major histocompatibility complex (MHC) molecules. Due to its limited host range, HCMV cannot be studied in animals. Thus, the in vivo importance of inhibiting antigen presentation for the establishment and maintenance of infection with HCMV is unknown. Rhesus cytomegalovirus (RhCMV) is an emerging animal model that shares many of the features of HCMV infection. The recent completion of the genomic sequence of RhCMV revealed a significant degree of homology to HCMV. Strikingly, RhCMV contains several genes with low homology to the HCMV US6 gene family of inhibitors of the MHC I antigen presentation pathway. Here, we examine whether the RhCMV US6 homologues (open reading frames Rh182, -184, -185, -186, -187, and -189) interfere with the MHC I antigen-processing pathway. We demonstrate that Rh182 and Rh189 function similarly to HCMV US2 and US11, respectively, mediating the proteasomal degradation of newly synthesized MHC I. The US3 homologue, Rh184, delayed MHC I maturation. Unlike US3, MHC I molecules eventually escaped retention by Rh184, so that steady-state surface levels of MHC I remained unchanged. Rh185 acted similarly to US6 and inhibited peptide transport by TAP and, consequently, peptide loading of MHC I molecules. Thus, despite relatively low sequence conservation, US6 family-related genes in RhCMV are functionally closely related to the conserved structural features of HCMV immunomodulators. The conservation of these mechanisms implies their importance for immune evasion in vivo, a question that can now be addressed experimentally.

The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence.

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.

Poxviruses and gamma-2 herpesviruses share the K3 family of viral immune evasion proteins that inhibit the surface expression of glycoproteins such as major histocompatibility complex class I (MHC-I), B7.2, ICAM-1, and CD95(Fas). K3 family proteins contain an amino-terminal PHD/LAP or RING-CH domain followed by two transmembrane domains. To examine whether human homologues are functionally related to the viral immunoevasins, we studied seven membrane-associated RING-CH (MARCH) proteins. All MARCH proteins located to subcellular membranes, and several MARCH proteins reduced surface levels of known substrates of the viral K3 family. Two closely related proteins, MARCH-IV and MARCH-IX, reduced surface expression of MHC-I molecules. In the presence of MARCH-IV or MARCH-IX, MHC-I was ubiquitinated and rapidly internalized by endocytosis, whereas MHC-I molecules lacking lysines in their cytoplasmic tail were resistant to downregulation. The amino-terminal regions containing the RING-CH domain of several MARCH proteins examined catalyzed multiubiquitin formation in vitro, suggesting that MARCH proteins are ubiquitin ligases. The functional similarity of the MARCH family and the K3 family suggests that the viral immune evasion proteins were derived from MARCH proteins, a novel family of transmembrane ubiquitin ligases that seems to target glycoproteins for lysosomal destruction via ubiquitination of the cytoplasmic tail.

Many viruses have developed mechanisms to escape the cellular immune response by inhibiting antigen presentation from major histocompatibility complex (MHC) molecules. Most of these immune escape mechanisms are highly host adapted and specific to a given virus species or family. Recent observations however, suggest that a conserved family of viral proteins is used by both gamma-2 herpesviruses and by poxviruses to downregulate MHC class I. In addition, other cell surface molecules involved in immune recognition by T cells and NK cells are also downregulated. Two open reading frames (ORFs), K3 and K5, of Kaposi's sarcoma associated virus (KSHV) and one ORFs, K3, of murine gamma herpesvirus 68 (MHV 68) inhibit surface expression of MHC I molecules. In cells transfected with KSHV-K3 and KSHV-K5, MHC I is rapidly endocytosed and degraded in lysosomes whereas in MHV 68-K3 transfected cells, MHC I is targeted for proteasomal degradation. The K3 and K5 genes display a characteristic conserved domain structure of an amino-terminal plant homeo domain/leukemia associated protein-zinc finger domain followed by two carboxyterminal transmembrane domains. Related proteins are not only found in other gamma-2 herpesviruses, but also in several poxviruses. Moreover, recent data suggest that the K3-related protein of myxoma virus also downregulates MHC I. The presence of similar genes in eukaryotic genomes further indicates that the viral ORFs were originally derived from host genes of as yet unknown function. The molecular mechanism of MHC I downregulation by this novel gene family is only poorly understood at present. However, several lines of evidence suggest that they might function as ubiquitin ligases that regulate the intracellular transport of transmembrane proteins through ubiquitination.

Herpes simplex virus serotype 1 (HSV-1) expresses an immediate-early protein, ICP47, that effectively blocks the major histocompatibility complex class I antigen presentation pathway. HSV-1 ICP47 (ICP47-1) binds with high affinity to the human transporter associated with antigen presentation (TAP) and blocks the binding of antigenic peptides. HSV type 2 (HSV-2) ICP47 (ICP47-2) has only 42% amino acid sequence identity with ICP47-1. Here, we compared the levels of inhibition of human and murine TAP, expressed in insect cell microsomes, by ICP47-1 and ICP47-2. Both proteins inhibited human TAP at similar concentrations, and the K(D) for ICP47-2 binding to human TAP was 4.8 x 10(-8) M, virtually identical to that measured for ICP47-1 (5.2 x 10(-8) M). There was some inhibition of murine TAP by both ICP47-2 and ICP47-1, but this inhibition was incomplete and only at ICP47 concentrations 50 to 100 times that required to inhibit human TAP. Lack of inhibition of murine TAP by ICP47-1 and ICP47-2 could be explained by an inability of both proteins to bind to murine TAP.

Human cytomegalovirus (HCMV) inhibits MHC class I antigen presentation by a sequential multistep process involving a family of unique short (US) region-encoded glycoproteins. US3 retains class I molecules, whereas US2 and US11 mediate the cytosolic degradation of heavy chains by the proteosomes. In US6-transfected cells, however, intracellular transport of class I molecules is impaired because of defective peptide translocation by transporters associated with antigen processing (TAP). Peptide transport is restored in HCMV mutants lacking US6. In contrast to the cytosolic herpes simplex virus protein ICP47, US6 interacts with TAP inside the endoplasmic reticulum lumen, as shown by US6 derivatives lacking the transmembrane and cytoplasmic domains and by the observation that US6 does not prevent peptides from binding to TAP. Thus, HCMV targets TAP for immune escape by a molecular mechanism different from that of herpes simplex virus.

An essential part of the immune response to viral infections is the recognition and elimination of infected cells by cytotoxic T lymphocytes. For this purpose a display mechanism has evolved which is present in almost all nucleated cells: the major histocompatibility complex class I antigen processing pathway. Both self and foreign antigens are degraded in the cytosol to peptides which are translocated into the endoplasmic reticulum where they are loaded onto MHC class I molecules. Pathogens living inside the cell are evolving under the constant selection pressure of such immune surveillance. As a result such infectious organisms have developed a variety of strategies to prevent their antigens from being presented. Since our understanding of the cell biology of antigen presentation has greatly advanced in recent years, it has now become possible to unravel several of the molecular mechanisms by which viruses interfere with MHC class I antigen presentation. Examples for the interference of viral molecules with components of the MHC class I pathway are presented in this review.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.