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.     

De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.     

Persistent activation of STAT3 by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castleman's disease. STAT3 has been shown to be important for the maintenance of primary effusion lymphoma cells in culture and is chronically activated in many tumor cell lines. However, little is known about the role of KSHV in the activation of STAT3 or the role of STAT3 in KS tumors. We demonstrate that STAT3 is activated by KSHV infection of endothelial cells, the KS tumor cell type, in a biphasic fashion. Viral binding and entry activate STAT3 in the first 2 h after infection, but this activation dissipates by 4 h postinfection. By 12 h after KSHV infection, concomitant with the expression of latent genes, STAT3 is once again activated, and this activation persists for as long as latent infection is maintained. Activated STAT3 translocates to the nucleus, where it can bind to STAT3-specific DNA elements and can activate STAT3-dependent promoter activity. Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. KSHV upregulates gp130 receptor expression, and both gp130 and JAK2 are required for the activation of STAT3. However, neither human nor viral interleukin-6 is required for STAT3 activation. Persistent activation of the oncogenic signal transducer, STAT3, by KSHV may play a critical role in the viral pathogenesis of Kaposi's sarcoma, as well as in primary effusion lymphomas.     

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

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

Kaposi's sarcoma-associated herpesvirus K3 utilizes the ubiquitin-proteasome system in routing class major histocompatibility complexes to late endocytic compartments

Human herpesvirus 8 (HHV8) downregulates major histocompatibility complex (MHC) class I complexes from the plasma membrane via two of its genes, K3 and K5. The N termini of K3 and K5 contain a plant homeodomain (PHD) predicted to be structurally similar to RING domains found in E3 ubiquitin ligases. In view of the importance of the ubiquitin-proteasome system in sorting within the endocytic pathway, we analyzed its role in downregulation of MHC class I complexes in cells expressing K3. Proteasome inhibitors as well as cysteine and aspartyl protease inhibitors stabilize MHC class I complexes in cells expressing K3. However, proteasome inhibitors differentially affect sorting of MHC class I complexes within the endocytic pathway and prevent their delivery to a dense endosomal compartment. In this compartment, the cytoplasmic tail of MHC class I complexes is cleaved by cysteine proteases. The complex is then cleaved within the plane of the membrane by an aspartyl protease, resulting in a soluble MHC class I fragment composed of the lumenal domain of the heavy chain, beta(2)-microglobulin (beta(2)m), and peptide. We conclude that K3 not only directs internalization, but also targets MHC class I complexes to a dense endocytic compartment on the way to lysosomes in a ubiquitin-proteasome-dependent manner.     

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

c-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells

During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3β1, αVβ3, and αVβ5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3β1, αVβ3, and x-CT receptors but not αVβ5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3β1 and αVβ3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MβCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3β1, -αVβ3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.     

Kaposi's sarcoma-associated herpesvirus infection promotes invasion of primary human umbilical vein endothelial cells by inducing matrix metalloproteinases

Matrix metalloproteinases (MMPs) play important roles in cancer invasion, angiogenesis, and inflammatory infiltration. Kaposi's sarcoma is a highly disseminated angiogenic tumor of proliferative endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we showed that KSHV infection increased the invasiveness of primary human umbilical vein endothelial cells (HUVEC) in a Matrigel-based cell invasion assay. KSHV-induced cell invasion was abolished by an inhibitor of MMPs, BB-94, and occurred in both autocrine- and paracrine-dependent fashions. Analysis by zymography and Western blotting showed that KSHV-infected HUVEC cultures had increased secretion of MMP-1, -2, and -9. KSHV increased the secretion of MMP-2 within 1 h following infection without upregulating its mRNA expression level. In contrast, the secretion of MMP-1 and -9 was not increased until 6 h after KSHV infection and was correlated with the upregulation of their mRNA expression levels. Promoter analysis by reporter assays and electrophoretic mobility shift assays identified an AP-1 cis-element as the dominant KSHV-responsive site in the MMP-1 promoter. Together, these results suggest that KSHV infection modulates the production of multiple MMPs to increase cell invasiveness and thus contributes to the pathogenesis of KSHV-induced malignancies.     

Kaposi's sarcoma-associated herpesvirus activation of vascular endothelial growth factor receptor 3 alters endothelial function and enhances infection

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8) is the etiologic agent of Kaposi's sarcoma, an endothelial neoplasm. This gamma-herpesvirus encodes for several unique proteins that alter target cell function, including the virion envelope-associated glycoprotein B (gB). Glycoprotein B has an RGD (Arg-Gly-Asp) motif at the extracellular amino terminus region and binds to the alpha3beta1 surface integrin, which enhances virus entry. We now report that gB can activate the vascular endothelial growth factor receptor 3 (VEGFR-3) on the surface of microvascular endothelial cells and trigger receptor signaling, which can modulate endothelial migration and proliferation. Furthermore, we observed that VEGFR-3 expression and activation enhance KSHV infection and participate in KSHV-mediated transformation. These functional changes in the endothelium may contribute to the pathogenesis of Kaposi's sarcoma and suggest that interventions that inhibit gB activation of VEGFR-3 could be useful in the treatment of this neoplasm.     

Pol-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells prior to Nef-mediated major histocompatibility complex class I downregulation

Effective, vaccine-induced CD8+ T-cell responses should recognize infected cells early enough to prevent production of progeny virions. We have recently shown that Gag-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells at 2 h postinfection, whereas Env-specific CD8+ T cells do not recognize infected cells until much later in infection. However, it remains unknown when other proteins present in the viral particle are presented to CD8+ T cells after infection. To address this issue, we explored CD8+ T-cell recognition of epitopes derived from two other relatively large virion proteins, Pol and Nef. Surprisingly, infected cells efficiently presented CD8+ T-cell epitopes from virion-derived Pol proteins within 2 h of infection. In contrast, Nef-specific CD8+ T cells did not recognize infected cells until 12 h postinfection. Additionally, we show that SIVmac239 Nef downregulated surface major histocompatibility complex class I (MHC-I) molecules beginning at 12 h postinfection, concomitant with presentation of Nef-derived CD8+ T-cell epitopes. Finally, Pol-specific CD8+ T cells eliminated infected cells as early as 6 h postinfection, well before MHC-I downregulation, suggesting a previously underappreciated antiviral role for Pol-specific CD8+ T cells.     

Productive lytic replication of a recombinant Kaposi's sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.