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The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19.Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow–derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1− CD127− CX3CR1− Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

Significance Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons., The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

To prevent accumulation of misfolded proteins in the endoplasmic reticulum, chaperones perform quality control on newly translated proteins and redirect misfolded proteins to the cytosol for degradation by the ubiquitin-proteasome system. This pathway is called ER-associated protein degradation (ERAD). The human cytomegalovirus protein US2 induces accelerated ERAD of HLA class I molecules to prevent immune recognition of infected cells by CD8+ T cells. Using US2-mediated HLA-I degradation as a model for ERAD, we performed a genome-wide CRISPR/Cas9 library screen to identify novel cellular factors associated with ERAD. Besides the identification of known players such as TRC8, p97, and UBE2G2, the ubiquitin-fold modifier1 (UFM1) pathway was found to affect degradation of HLA-I. UFMylation is a post-translational modification resembling ubiquitination. Whereas we observe ubiquitination of HLA-I, no UFMylation was detected on HLA-I or several other proteins involved in degradation of HLA-I, suggesting that the UFM1 pathway impacts ERAD in a different manner than ubiquitin. Interference with the UFM1 pathway seems to specifically inhibit the ER-to-cytosol dislocation of HLA-I. In the absence of detectable UFMylation of HLA-I, UFM1 may contribute to US2-mediated HLA-I degradation by misdirecting protein sorting indirectly. Mass spectrometry analysis of US2-expressing cells showed that ribosomal proteins are a major class of proteins undergoing extensive UFMylation, the role of these changes in protein degradation may be indirect and remains to be established.

MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the ‘bulge’ sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

Viruses may interfere with the MHC class I antigen presentation pathway in order to avoid CD8+ T cell-mediated immunity. A key target within this pathway is the peptide transporter TAP. This transporter plays a central role in MHC class I-mediated peptide presentation of endogenous antigens. In addition, TAP plays a role in antigen cross-presentation of exogenously derived antigens by dendritic cells (DCs). In this study, a soluble form of the cowpox virus TAP inhibitor CPXV012 is synthesized for exogenous delivery into the antigen cross-presentation route of human monocyte-derived (mo)DCs. We show that soluble CPXV012 localizes to TAP+ compartments that carry internalized antigen and is a potent inhibitor of antigen cross-presentation. CPXV012 stimulates the prolonged deposition of antigen fragments in storage compartments of moDCs, as a result of reduced endosomal acidification and reduced antigen proteolysis when soluble CPXV012 is present. Thus, a dual function can be proposed for CPXV012: inhibition of TAP-mediated peptide transport and inhibition of endosomal antigen degradation. We propose this second function for soluble CPXV012 can serve to interfere with antigen cross-presentation in a peptide transport-independent manner.

Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN–induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.

Summary Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection., Graphical Abstract, Highlights • The RNF185 ubiquitin ligase, Membralin, and TMUB1/2 assemble into an ERAD complex • RNF185/Membralin complex targets membrane proteins, including CYP51A1 and TMUB2 • RNF185/Membralin and TEB4 ERAD complexes recognize distinct substrate features • TEB4 ERAD complex recognizes substrates through their transmembrane domain, Membrane proteins are diverse, and the components involved in their quality control in each case are unclear. By comparing the degradation of two short-lived ER proteins, van de Weijer et al. identified an ERAD complex composed of RNF185, TMUBs, and Membralin, a membrane protein essential to neuronal function.

MHC class I molecules are an important component of the cell-mediated immune defense, presenting peptides to surveilling CD8

Newly translated proteins enter the ER through the SEC61 complex, via either co- or post-translational translocation. In mammalian cells, few substrates of post-translational SEC62- and SEC63-dependent translocation have been described. Here, we targeted all components of the SEC61/62/63 complex by CRISPR/Cas9, creating knock-outs or mutants of the individual subunits of the complex. We show that functionality of the human cytomegalovirus protein US2, which is an unusual translocation substrate with a low-hydrophobicity signal peptide, is dependent on expression of not only SEC61α, -β, and -γ, but also SEC62 and SEC63, suggesting that US2 may be a substrate for post-translational translocation. This phenotype is specific to the US2 signal peptide.

T-cell mediated adaptive immunity against viruses relies on recognition of virus-derived peptides by CD4(+) and CD8(+) T cells. Detection of pathogen-derived peptide-MHC-I complexes triggers CD8(+) T cells to eliminate the infected cells. Viruses have evolved several mechanisms to avoid recognition, many of which target the MHC-I antigen-processing pathway. While many immune evasion strategies have been described in the context of herpesvirus infections, it is becoming clear that this 'disguise' ability is more widespread. Here, we address recent findings in viral evasion of the MHC-I antigen presentation pathway and the impact on CD8(+) T cell responses.

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.

Contains fulltext : 205154.pdf (Publisher’s version ) (Closed access) Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24delta) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

MHC class I molecules are an important component of the cell-mediated immune defense, presenting peptides to surveilling CD8+ cytotoxic T cells. During viral infection, MHC class I molecules carry and display viral peptides at the cell surface. CD8+ T cells that recognize these peptides will eliminate the virus-infected cells. Viruses counteract this highly sophisticated host detection system by downregulating cell surface expression of MHC class I molecules. In this chapter, we describe a flow cytometry-based method that can be used for the identification of viral gene products potentially responsible for evasion from MHC class I-restricted antigen presentation. The gene(s) of interest are expressed constitutively through lentiviral transduction of cells. Subsequently, MHC I surface expression is monitored using MHC class I-specific antibodies. Once the viral gene product responsible for MHC I downregulation has been identified, the same cells can be used to elucidate the mechanism of action. The stage at which interference with antigen processing occurs can be identified using specific assays. An essential step frequently targeted by viruses is the translocation of peptides into the ER by the transporter associated with antigen processing, TAP. TAP function can be measured using a highly specific in vitro assay involving flow cytometric evaluation of the import of a fluorescent peptide substrate. The protocol described in this chapter enables the identification of virus-encoded MHC class I inhibitors that hinder antigen processing and presentation. Subsequently, their mechanism of action can be unraveled; this knowledge may help to rectify their actions.

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2 and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, suggesting that TMED10 regulates actin cytoskeleton remodelling necessary for virus infection. Importance Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells, and are used as vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knock-out library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, modulates the host cell membrane to facilitate virus uptake.

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

The MHC class I antigen presentation pathway enables cells infected with intracellular pathogens to signal the presence of the invader to the immune system. Cytotoxic T lymphocytes are able to eliminate the infected cells through recognition of pathogen-derived peptides presented by MHC class I molecules at the cell surface. In the course of evolution, many viruses have acquired inhibitors that target essential stages of the MHC class I antigen presentation pathway. Studies on these immune evasion proteins reveal fascinating strategies used by viruses to elude the immune system. Viral immunoevasins also constitute great research tools that facilitate functional studies on the MHC class I antigen presentation pathway, allowing the investigation of less well understood routes, such as TAP-independent antigen presentation and cross-presentation of exogenous proteins. Viral immunoevasins have also helped to unravel more general cellular processes. For instance, basic principles of ER-associated protein degradation via the ubiquitin-proteasome pathway have been resolved using virus-induced degradation of MHC class I as a model. This review highlights how viral immunoevasins have increased our understanding of MHC class I-restricted antigen presentation.

Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2- induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system.

Unraveling the molecular arms race between virus and host has been taken to a new level. A cryo-EM study reveals in unprecedented detail how the herpesvirus immune-evasion protein ICP47 inhibits the peptide transporter TAP.

Misfolded MHC class I heavy chains (MHC I HCs) are targeted for endoplasmic reticulum (ER)-associated degradation (ERAD) by the ubiquitin E3 ligase HRD1, and E2 ubiquitin conjugating enzyme UBE2J1, and represent one of the few known endogenous ERAD substrates. The mechanism by which misfolded proteins are dislocated across the ER membrane into the cytosol is unclear. Here, we investigate the requirements for MHC I ubiquitination and degradation and show that endogenous misfolded MHC I HCs are recognized in the ER lumen by EDEM1 in a glycan-dependent manner and targeted to the core SEL1L/HRD1/UBE2J1 complex. A soluble MHC I HC lacking its transmembrane domain and cytosolic tail uses the same ERAD components and is degraded as efficiently as wild-type MHC I. Unexpectedly, HRD1-dependent polyubiquitination is preferentially targeted to the ER luminal domain of full-length MHC I HCs, despite the presence of an exposed cytosolic C-terminal tail. MHC I luminal domain ubiquitination occurs before p97 ATPase-mediated extraction from the ER membrane and can be targeted to nonlysine, as well as lysine, residues. A subset of integral membrane proteins, therefore, requires an early dislocation event to expose part of their luminal domain to the cytosol, before HRD1-mediated polyubiquitination and dislocation.

Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human beta cells to express herpesvirus-encoded immune-evasion proteins, "immunevasins." The capacity of immunevasins to protect beta cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human beta cells without impairing their function. Using a novel beta-cell-specific reporter gene assay, we show that autoreactive cytotoxic CD8(+) T-cell clones isolated from patients with recent onset diabetes selectively destroyed human beta cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with beta-cell-specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human beta cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.

HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.

Coevolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells and viral ploys to escape immune surveillance. Among the immune-evasion strategies used by the lymphocryptovirus (γ1-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally encoded G protein–coupled receptor BILF1, thereby impeding presentation of viral Ags and cytotoxic T cell recognition of the infected cell. In this study, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA-A and -B types and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the process by showing that the cytoplasmic C-terminal tail of EBV BILF1 is required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation, in turn, is conferred by specific residues in the intracellular tail of the HLA class I H chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. Although the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. Therefore, this study furthers our knowledge of the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses.

Background Epstein-Barr virus (EBV) establishes lifelong infections in its human host. The virus is associated with a broad range of malignancies of lymphoid and epithelial origin, including Burkitt’s lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. During the latent phase of its life cycle, EBV expresses more than 40 mature miRNAs that are highly abundant in tumor cells and may contribute to oncogenesis. Although multiple studies have assessed the relative expression profiles of EBV miRNAs in tumor cells, data linking these expression levels to functional target knockdown are mostly lacking. Therefore we set out to systematically assess the EBV miRNA expression levels in EBV+ tumor cell lines, and correlate this to their functional silencing capacity in these cells. Results We provide comprehensive EBV miRNA expression profiles of the EBV+ cell lines C666-1 (nasopharyngeal carcinoma), SNU-719 (gastric carcinoma), Jijoye (Burkitt’s lymphoma), and AKBM (Burkitt’s lymphoma) and of EBV− cells ectopically expressing the BART miRNA cluster. By deep sequencing the small RNA population and conducting miRNA-reporter experiments to assay miRNA potency, we were able to compare the expression profiles of the EBV miRNAs with their functional silencing efficacy. We observe a strong correlation between miRNA expression levels and functional miRNA activity. There is large variation in expression levels between EBV miRNAs in a given cell line, whereas the relative expression profiles are well maintained between cell lines. Furthermore, we show that miRNA arm selection bias is less pronounced for gamma-herpesvirus miRNAs than for human miRNAs. Conclusion We provide an in depth assessment of the expression levels and silencing activity of all EBV miRNAs in B- and epithelial cell lines of different latency stages. Our data show a good correlation between relative EBV miRNA expression levels and silencing capacity, and suggest preferential processing of particular EBV miRNAs irrespective of cell-type. In addition to encoding the largest number of precursor miRNAs of all human herpesviruses, EBV expresses many miRNAs precursors that yield two functional miRNA strands, rather than one guide strand and a non-functional passenger strand. This reduced strand bias may increase the size of the EBV miRNA targetome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2978-6) contains supplementary material, which is available to authorized users.

Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle., Author Summary The human herpesvirus Epstein-Barr virus (EBV) is an important human pathogen involved in infectious mononucleosis and several malignant tumors, including lymphomas in the immunosuppressed. Upon primary infection, a balance between virus and host is established, to which EBV’s capacity to dodge T cell-mediated attack contributes. Here we identify the late protein EBV gp150 as a novel immunoevasin, frustrating antigen presentation by HLA class I, class II, and CD1d molecules. EBV gp150’s many sialoglycans create a shield impeding surface detection of presented antigen. Interestingly, exploiting glycan shielding as a mechanism to mask surface exposed proteins on infected cells could permit EBV to additionally modulate other aspects of host antiviral defense. B cells producing wild-type EBV escaped immune recognition more efficiently than those infected with a gp150-null virus, pointing towards a role for gp150 in natural infection. Our results reveal a novel, broadly active strategy by which a herpesvirus glycoprotein, EBV gp150, blocks antigen presentation to T cells through glycan shielding, a new paradigm in herpesvirus immune evasion.

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the t ransporter associated with a ntigen p rocessing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.

Upon infecting a host, viruses are confronted by a coordinated and multi-faceted immune response. Indeed, evolutionary combat between virus and host has contributed signally to the host's development of a formidable innate and adaptive immune defense arsenal, and to the virus' acquisition of effective means to evade it. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, which they detect through recognition of virus-derived peptides displayed at the cell surface in the context of MHC class I molecules. This highly sensitive recognition system is a prime target for immune evasion strategies deployed by many viruses, particularly large DNA viruses such as herpesviruses and poxviruses. Elucidation of the mode of action of the immune evasion proteins encoded by these viruses has not only provided new insights into viral pathogenesis, but has also led to the discovery of hitherto unknown cell biological and immunological phenomena. Moreover, viral immune evasion proteins constitute extremely useful tools to block defined stages of the MHC class I presentation pathway, not only for research purposes, but also for clinical applications.

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu439, which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu439 by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

During HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. This study explores an alternative HLA class II presentation pathway in leukemic blasts that involves proteasome and transporter associated with antigen processing (TAP)-dependent peptide loading. Although HLA-DR did associate with Ii, Ii silencing in the human class II-associated invariant chain peptide (CLIP)-negative KG-1 myeloid leukemic cell line did not affect total and plasma membrane expression levels of HLA-DR, as determined by western blotting and flow cytometry. Since HLA-DR expression does require peptide binding, we examined the role of endogenous antigen-processing machinery in HLA-DR presentation by CLIP− leukemic blasts. The suppression of proteasome and TAP function using various inhibitors resulted in decreased HLA-DR levels in both CLIP− KG-1 and ME-1 blasts. Simultaneous inhibition of TAP and Ii completely down-modulated the expression of HLA-DR, demonstrating that together these molecules form the key mediators of HLA class II antigen presentation in leukemic blasts. By the use of a proteasome- and TAP-dependent pathway for HLA class II antigen presentation, CLIP− leukemic blasts might be able to present a broad range of endogenous leukemia-associated peptides via HLA class II to activate leukemia-specific CD4+ T cells. Electronic supplementary material The online version of this article (doi:10.1007/s00262-010-0908-z) contains supplementary material, which is available to authorized users.

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.

Summary Cowpox virus encodes an extensive array of putative immunomodulatory proteins, likely contributing to its wide host range, which includes zoonotic infections in humans. Unlike Vaccinia virus, cowpox virus prevents stimulation of CD8 + T cells, a block that correlated with retention of MHC class I in the endoplasmic reticulum by the cowpox virus protein CPXV203. However, deletion of CPXV203 did not restore MHC class I transport or T cell stimulation. Here, we demonstrate the contribution of an additional viral protein, CPXV12, which interferes with MHC class I/peptide complex formation by inhibiting peptide translocation by the transporter associated with antigen processing (TAP). Importantly, human and mouse MHC class I transport and T cell stimulation was restored upon deletion of both CPXV12 and CPXV203 , suggesting that these unrelated proteins independently mediate T cell evasion in multiple hosts. CPXV12 is a truncated version of a putative NK cell ligand, indicating that poxviral gene fragments can encode new, unexpected functions.

Cross-presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ-resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross-presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross-presenting circulating soluble antigen ex vivo as DCs at a per-cell basis. However, antigen uptake in vivo was 100-fold more pronounced in LSECs, indicating distinct mechanisms of cross-presentation. In contrast to mannose-receptor–mediated antigen uptake and routing into stable endosomes dedicated to cross-presentation in DCs, we observed distinct antigen-uptake and endosomal routing with high antigen turnover in LSECs that resulted in short-lived cross-presentation. Receptor-mediated endocytosis did not always lead to cross-presentation, because immune-complexed antigen taken up by the Fc-receptor was not cross-presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity. Conclusion: These results provide a mechanistic explanation how organ-resident LSECs accommodate continuous scavenger function with the capacity to cross-present circulating antigens using distinct kinetics and dynamics of antigen-uptake, routing and cross-presentation compared to DCs. (HEPATOLOGY 2009.)

EBV persists for life in the human host while facing vigorous antiviral responses that are induced upon primary infection. This persistence supports the idea that herpesviruses have acquired dedicated functions to avoid immune elimination. The recently identified EBV gene product BNLF2a blocks TAP. As a result, reduced amounts of peptides are transported by TAP from the cytoplasm into the endoplasmic reticulum (ER) lumen for binding to newly synthesized HLA class I molecules. Thus, BNLF2a perturbs detection by cytotoxic T cells. The 60-aa-long BNLF2a protein prevents the binding of both peptides and ATP to TAP, yet further mechanistic insight is, to date, lacking. In this study, we report that EBV BNLF2a represents a membrane-associated protein that colocalizes with its target TAP in subcellular compartments, primarily the ER. In cells devoid of TAP, expression levels of BNLF2a protein are greatly diminished, while ER localization of the remaining BNLF2a is retained. For interactions of BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Importantly, we now show that in B cells supporting EBV lytic replication, the BNLF2a protein is expressed early in infection, colocalizing and associating with the peptide-loading complex. These results imply that, during productive EBV infection, BNLF2a contributes to TAP inhibition and surface HLA class I down-regulation. In this way, EBV BNLF2a-mediated evasion from HLA class I-restricted T cell immunity contributes to creating a window for undetected virus production.

Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase inhibitor.

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.

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen ( MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.

The mechanism by which substrates for endoplasmic reticulum–associated degradation are retrotranslocated to the cytosol remains largely unknown, although ubiquitination is known to play a key role. The mouse γ-herpesvirus protein mK3 is a viral RING-CH–type E3 ligase that specifically targets nascent major histocompatibility complex I heavy chain (HC) for degradation, thus blocking the immune detection of virus-infected cells. To address the question of how HC is retrotranslocated and what role mK3 ligase plays in this action, we investigated ubiquitin conjugation sites on HC using mutagenesis and biochemistry approaches. In total, our data demonstrate that mK3-mediated ubiquitination can occur via serine, threonine, or lysine residues on the HC tail, each of which is sufficient to induce the rapid degradation of HC. Given that mK3 has numerous cellular and viral homologues, it will be of considerable interest to determine the pervasiveness of this novel mechanism of ubiquitination.

Epstein-Bar virus (EBV) is widespread within the human population with over 90% of adults being infected. In response to primary EBV infection, the host mounts an antiviral immune response comprising both innate and adaptive effector functions. Although the immune system can control EBV infection to a large extent, the virus is not cleared. Instead, EBV establishes a latent infection in B lymphocytes characterized by limited viral gene expression. For the production of new viral progeny, EBV reactivates from these latently infected cells. During the productive phase of infection, a repertoire of over 80 EBV gene products is expressed, presenting a vast number of viral antigens to the primed immune system. In particular the EBV-specific CD4+ and CD8+ memory T lymphocytes can respond within hours, potentially destroying the virus-producing cells before viral replication is completed and viral particles have been released. Preceding the adaptive immune response, potent innate immune mechanisms provide a first line of defense during primary and recurrent infections. In spite of this broad range of antiviral immune effector mechanisms, EBV persists for life and continues to replicate. Studies performed over the past decades have revealed a wide array of viral gene products interfering with both innate and adaptive immunity. These include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. The current review presents an overview of the evasion strategies that are employed by EBV to facilitate immune escape during latency and productive infection. These evasion mechanisms may also compromise the elimination of EBV-transformed cells, and thus contribute to malignancies associated with EBV infection.

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses., Author Summary Natural killer (NK) cells recognize and kill infected cells without prior antigenic stimulation, and thus provide an important early defense against virus infection. NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. Inhibitory KIRs normally suppress NK cell responses through interactions with their MHC class I ligands on the surface of healthy cells. However, when these interactions are perturbed, this inhibition is lost resulting in NK cell activation and killing of the target cell. We investigated the functional implications of simian immunodeficiency virus (SIV) peptides bound by a common MHC class I molecule in the rhesus macaque that stabilize or disrupt binding to an inhibitory KIR. Whereas SIV peptides that stabilized KIR-MHC class I binding suppressed NK cell activation, peptides that disrupted this interaction did not and resulted in NK cell lysis. These findings demonstrate that viral peptides can modulate NK cell responses through KIR-MHC class I interactions, and are consistent with the possibility that human and simian immunodeficiency viruses may acquire changes in epitopes that increase the binding of MHC class I ligands to inhibitory KIRs as a mechanism to suppress NK cell responses.

Surface MHC class I molecules serve important immune functions as ligands for both T and NK cell receptors for the elimination of infected and malignant cells. In order to reach the cell surface, MHC class I molecules have to fold properly and form trimers consisting of a heavy chain (HC), a beta2-microglobulin light chain and an 8-10-mer peptide. A panel of ER chaperones facilitates the folding and assembly process. Incorrectly assembled or folded MHC class I HCs are detected by the ER quality-control system and transported to the cytosol for degradation by proteasomes. In human cytomegalovirus-infected cells, two viral proteins are synthesized, US2 and US11, which target MHC class I HCs for proteasomal degradation. It is unknown at which stage of MHC class I folding and complex formation US2 and US11 come into play. In addition, it is unclear if the disposal takes place via the same pathway through which proteins are removed that fail to pass ER quality control. In this study, we show with a beta2m-deficient cell line that US2 and US11 both target unassembled HCs for degradation. This suggests that US2 and US11 both act at an early stage of MHC class I complex formation. In addition, our data indicate that US11-mediated degradation involves mechanisms that are similar to those normally used to remove terminally misfolded HCs.