Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.     

Intracellular Kaposi's sarcoma-associated herpesvirus load determines early loss of immune synapse components

Lifelong infection is a hallmark of all herpesviruses, and their survival depends on countering host immune defenses. The human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of proteins that contribute to immune evasion, including modulator of immune recognition 2 (MIR2), an E3 ubiquitin ligase. Exogenously expressed MIR2 downregulates the surface expression of several immune synapse proteins, including major histocompatibility complex (MHC) class 1, ICAM-1 (CD54), and PECAM (CD31). Although immunofluorescence assays detect this lytic gene in only 1 to 5% of cells within infected cultures, we have found that de novo infection of naive cells leads to the downregulation of these immune synapse components in a major proportion of the population. Investigating the possibility that low levels of MIR2 are responsible for this downregulation in the context of viral infection, we found that MIR2 transduction recapitulated the patterns of surface downregulation following de novo infection and that both MIR2 promoter activation, MIR2 expression level, and immune synapse component downregulation were proportional to the concentration of KSHV added to the culture. Additionally, MIR2-specific small interfering RNA reversed the downregulation effects. Finally, using a sensitive, high-throughput assay to detect levels of the virus in individual cells, we also observed that downregulation of MHC class I and ICAM-1 correlated with intracellular viral load. Together, these results suggest that the effects of MIR2 are gene dosage dependent and that low levels of this viral protein contribute to the widespread downregulation of immune-modulating cell surface proteins during the initial stages of KSHV infection.     

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.     

Multiple endocytic trafficking pathways of MHC class I molecules induced by a Herpesvirus protein

The K3 protein of a human tumor-inducing herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), down-regulates major histocompatibility complex (MHC) class I surface expression by increasing the rate of endocytosis. In this report, we demonstrate that the internalization of MHC class I by the K3 protein is the result of multiple, consecutive trafficking pathways that accelerate the endocytosis of class I molecules, redirect them to the trans-Golgi network (TGN), and target MHC class I to the lysosomal compartment. Remarkably, these actions of K3 are functionally and genetically separable; the N-terminal zinc finger motif and the central sorting motif are involved in triggering internalization of MHC class I molecules and redirecting them to the TGN. Subsequently, the C-terminal diacidic cluster region of K3 is engaged in targeting MHC class I molecules to the lysosomal compartment. These results demonstrate a novel trafficking mechanism of MHC class I molecules induced by KSHV K3, which ensures viral escape from host immune effector recognition.     

Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (～5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.     

Upregulation of the TLR3 pathway by Kaposi's sarcoma-associated herpesvirus during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several different human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV establishes lifelong latency in the host and modulates the host immune response. Innate immunity is critical for controlling de novo viral infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. In particular, TLR3 has been implicated in RNA virus recognition. Currently, there is no information regarding how KSHV infection modulates any TLR pathway. We report the first evidence that KSHV upregulates TLR3 expression in human monocytes during primary infection. This is also the first demonstration of a human DNA tumor virus upregulating TLR3, a TLR that thus far has been associated with the recognition of RNA viruses. We found that KSHV upregulates the TLR3 pathway and induces TLR3-specific cytokines and chemokines, including beta 1 interferon (IFN-beta1) and CXCL10 (IP-10). Small interfering RNAs directed against TLR3 greatly reduced the ability of KSHV to upregulate IFN-beta1 and CXCL10 upon infection.     

The MARCH family E3 ubiquitin ligase K5 alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation

Kaposi's sarcoma (KS) lesions are complex mixtures of KS-associated herpesvirus (KSHV)-infected spindle and inflammatory cells. In order to survive the host immune responses, KSHV encodes a number of immunomodulatory proteins, including the E3 ubiquitin ligase K5. In exploring the role of this viral protein in monocytes, we made the surprising discovery that in addition to a potential role in down regulation of immune responses, K5 also contributes to increased proliferation and alters cellular metabolism. This ubiquitin ligase increases aerobic glycolysis and lactate production through modulation of cellular growth factor-binding receptor tyrosine kinase endocytosis, increasing the sensitivity of cells to autocrine and paracrine factors. This leads to an altered pattern of cellular phosphorylation, increases in Akt activation and a longer duration of Erk1/2 phosphorylation. Overall, we believe this to be the first report of a virally-encoded ubiquitin ligase potentially contributing to oncogenesis through alterations in growth factor signaling cascades and opens a new avenue of research in K5 biology.     

The intertransmembrane region of Kaposi's sarcoma-associated herpesvirus modulator of immune recognition 2 contributes to B7-2 downregulation

Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor virus, encodes two homologous membrane-associated E3 ubiquitin ligases, modulator of immune recognition 1 (MIR1) and MIR2, to evade host immunity. Both MIR1 and MIR2 downregulate the surface expression of major histocompatibility complex class I (MHC I) molecules through ubiquitin-mediated endocytosis followed by lysosomal degradation. Since MIR2 additionally downregulates a costimulatory molecule (B7-2) and an integrin ligand (intercellular adhesion molecule 1 [ICAM-1]), MIR2 is thought to be a more important molecule for immune evasion than MIR1; however, the molecular basis of the MIR2 substrate specificity remains unclear. To address this issue, we determined which regions of B7-2 and MIR2 are required for MIR2-mediated B7-2 downregulation. Experiments with chimeras made by swapping domains between human B7-2 and CD8α, a non-MIR2 substrate, and between MIR1 and MIR2 demonstrated a significant contribution of the juxtamembrane (JM) region of B7-2 and the intertransmembrane (ITM) region of MIR2 to MIR2-mediated downregulation. Structure prediction and mutagenesis analyses indicate that Phe119 and Ser120 in the MIR2 ITM region and Asp244 in the B7-2 JM region contribute to the recognition of B7-2 by MIR2. This finding provides new insight into the molecular basis of substrate recognition by MIR family members.     

The RING-CH Ligase K5 Antagonizes Restriction of KSHV and HIV-1 Particle Release by Mediating Ubiquitin-Dependent Endosomal Degradation of Tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi''s sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.     

Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposi's sarcoma-associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3-associated class I by lysine-63-linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine-63-linked polyubiquitination requires both UbcH5b/c and Ubc13-conjugating enzymes for initiating mono- and subsequent polyubiquitination of class I, and the clathrin-dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine-63-linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.     

Surface downregulation of major histocompatibility complex class I,PE-CAM,and ICAM-1 following de novo infection of endothelial cells with Kaposi's sarcoma-associated herpesvirus

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.     

Kaposi's sarcoma-associated herpesvirus cytotoxic T lymphocytes recognize and target Darwinian positively selected autologous K1 epitopes

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma (KS) and certain lymphoproliferations particularly in the context of human immunodeficiency virus (HIV) type 1-induced immunosuppression. The introduction of effective therapies to treat HIV has led to a decline in the incidence of KS, suggesting that immune responses may play a role in controlling KSHV infection and pathogenesis. Cytotoxic-T-lymphocyte (CTL) activity against KSHV proteins has been demonstrated; however, the identification of KSHV CTL epitopes remains elusive and problematic. Although the herpesvirus genomic layout is generally conserved, KSHV encodes a unique hypervariable protein, K1, with intense biological selection pressure at specific amino acid sites. To investigate whether this variability is partly driven by cellular immunity, we designed K1 peptides that match only the unique viral sequence for every individual studied here (autologous peptides). We identified functional CTL epitopes within K1's most variable areas, and we show that a given individual responds only to autologous peptides and not to peptides from other individuals. Furthermore, these epitopes are highly conserved sequences within KSHV isolates from a specific strain but are not conserved between different strains. We conclude that CTL recognition contributes to K1, and therefore to KSHV, evolution.     

Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.