-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2013; 8(2):e58056. · 4.09 Impact Factor
-
Infectious Agents and Cancer 05/2012; 5:1-2.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS Pathogens 04/2011; 7(4):e1001331. · 9.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Among pregnant women, acquired viral infections with a concurrent bacterial infection is a detrimental factor associated to poor prognosis. We evaluate the effect of a viral infection that does not lead to pre-term labor on the response to low doses of lipopolysaccharide (LPS). Our objectives were (i) to characterize the effect of a viral infection concurrent with exposure to microbial products on pregnancy outcome and (ii) to characterize the placental and fetal immune responses to the viral sensitization to LPS.
C57B/6 wild-type mice were injected with murine gammaherpesvirus 68 (MHV68) at E8.5. Either PBS or LPS was injected i.p. at E15.5. Pregnancy outcome and cytokine/chemokine profile from implantation sites were analyzed by multiplex.
LPS treatment of MHV-68-infected animals induced pre-term delivery and fetal death in 100% of the mice. Pre-term labor was characterized by a upregulation of pro-inflammatory cytokines and chemokines in both placenta and decidua. Similar profiles were observed from MHV-68-infected human primary trophoblast and trophoblast cell lines in response to LPS.
We describe for the first time that a sub-clinical viral infection in pregnant mice might sensitize to a bacterial infection leading to pre-term delivery. We propose the 'Double Hit Hypothesis' where the presence of a viral infection enhances the effect of bacterial products during pregnancy leading not only to pre-term labor but likely larger adverse outcomes.
American Journal Of Reproductive Immunology 02/2011; 65(2):110-7. · 2.17 Impact Factor
-
Ingrid Cardenas, Robert E Means,
Paulomi Aldo,
Kaori Koga,
Sabine M Lang,
Carmen J Booth,
Carmen Booth,
Alejandro Manzur,
Enrique Oyarzun,
Roberto Romero,
Gil Mor
[show abstract]
[hide abstract]
ABSTRACT: Pandemics pose a more significant threat to pregnant women than to the nonpregnant population and may have a detrimental effect on the well being of the fetus. We have developed an animal model to evaluate the consequences of a viral infection characterized by lack of fetal transmission. The experiments described in this work show that viral infection of the placenta can elicit a fetal inflammatory response that, in turn, can cause organ damage and potentially downstream developmental deficiencies. Furthermore, we demonstrate that viral infection of the placenta may sensitize the pregnant mother to bacterial products and promote preterm labor. It is critical to take into consideration the fact that during pregnancy it is not only the maternal immune system responding, but also the fetal/placental unit. Our results further support the immunological role of the placenta and the fetus affecting the global response of the mother to microbial infections. This is relevant for making decisions associated with treatment and prevention during pandemics.
The Journal of Immunology 07/2010; 185(2):1248-57. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Retroperitoneal fibromatosis-associated herpesvirus (RFHV) is a gamma-herpesvirus of macaques that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). Herein, we present characterization of the K3 gene from RFHV, a homologue of the KSHV K3 and K5 genes. Like the KSHV proteins, kK3 and kK5, the rfK3 protein decreases cell surface MHC I levels. Similar to kK5, rfK3 also modulates ICAM-1, but not another kK5 target, B7.2. Inhibitors of dynamin or mutations in the rfK3 RING-CH E3 ubiquitin ligase domain block cellular target regulation, implicating a ubiquitin-dependent, endocytosis-mediated mechanism for target down regulation and degradation. Overall, this manuscript presents the first characterization of a non-human primate virus MARCH family E3 ubiquitin ligase contributing important information about the evolution of immune avoidance strategies in primate viruses and paving the way for an animal model examining the importance of kK3 and kK5 in vivo.
Virology 03/2010; 398(2):214-23. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Rhesus monkey rhadinovirus (RRV) is highly related to Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus etiologically-linked with several cancers. Glycoprotein B (gB) homologues are encoded by all herpesviruses and play a role in virus attachment, entry, and in egress. We have found that RRV gB, like KSHV gB, is cleaved at a consensus furin cleavage site and is modified by both N-linked and O-linked glycosylation. Mutagenesis of three tyrosine- based trafficking motifs, a diacidic tyrosine motif, and a di-lucine motif in the cytoplasmic region revealed a role for these sequences in both ER export and endocytosis from the plasma membrane. These experiments provide a basis for further experiments looking at gB incorporation and role in gamma-herpesvirus assembly and egress.
Virology 03/2010; 398(2):233-42. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms.
Journal of Virology 07/2007; 81(12):6573-83. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The human γ-HVs are able to establish a lifelong, persistent infection that is largely clinically inapparent within the immunocompetent host. However, when these viruses are not kept in check, a variety of lymphoproliferative and neoplastic disorders result that will be detailed elsewhere within this volume. In brief, for HHV-8, also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), these neoplasias include Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). HHV-4, or Epstein–Barr virus (EBV), has been etiologically associated with infectious mononucleosis, Burkitt’s lymphoma, nasopharyngeal carcinoma (NPC), Hodgkin’s disease, hemophagocytic lymphohistiocytosis syndrome and some gastric cancers. Through coevolution with their hosts, these viruses have acquired a number of genes that act to set a fine balance between the uncontrolled, virally driven cellular proliferation seen in the immunocompromised host and complete elimination of infected cells by the immune responses. Several of these gene products cause selective suppression of normal immune system functioning and allow for an apathogenic, persistent infection.
01/2007; , ISBN: 9780521827140
-
[show abstract]
[hide abstract]
ABSTRACT: Epstein-Barr virus (EBV) EBNA2 and Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) are recruited to their responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jkappa. In particular, RTA and EBNA2 interactions with RBP-Jkappa are essential for the lytic replication of KSHV and expression of B-cell activation markers CD21 and CD23a, respectively. Here, we demonstrate that like EBV EBNA2, KSHV RTA strongly induces CD21 and CD23a expression through RBP-Jkappa binding sites in the first intron of CD21 and in the CD23a core promoter, respectively. However, unlike EBV EBNA2, which alters immunoglobulin mu (Igmu) and c-myc gene expression, RTA did not affect Igmu and c-myc expression, indicating that KSHV RTA targets the Notch signal transduction pathway in a manner similar to but distinct from that of EBV EBNA2. Furthermore, RTA-induced expression of CD21 glycoprotein, which is an EBV receptor, efficiently facilitated EBV infection. In addition, RTA-induced CD23 glycoprotein underwent proteolysis and gave rise to soluble CD23 (sCD23) molecules in B lymphocytes and KSHV-infected primary effusion lymphocytes. sCD23 then stimulated primary human lymphocytes. These results demonstrate that cellular CD21 and CD23a are common targets for B lymphotropic gammaherpesviruses and that KSHV RTA regulates RBP-Jkappa-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.
Journal of Virology 05/2005; 79(8):4651-63. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Upon viral infection, the major defense mounted by the host immune system is the activation of the interferon (IFN)-mediated antiviral pathway. In order to complete their life cycle, viruses that are obligatory intracellular parasites must modulate the host IFN-mediated immune response. Murine gammaherpesvirus 68 (gammaHV68) infects a wide range of cell types and establishes latent infections in mice. Here we demonstrate that the gammaHV68 latency-associated M2 protein has a cell-type-dependent localization pattern: M2 is present in the cytoplasm and plasma membrane in lymphocytes, whereas it is present primarily in the nucleus in epithelial and fibroblast cells. A mutational analysis indicated that the internal positively charged amino acids of M2 are required for its nuclear localization in fibroblasts. Purification of the M2 complex showed that M2 specifically interacts with the cellular p32 acidic protein through its central positively charged region and that this interaction recruits the cellular p32 protein to the nucleus in fibroblasts. Regardless of its localization, M2 expression effectively induced the downregulation of STAT1 and/or STAT2 in both A20 B lymphocytes and NIH 3T3 fibroblasts, resulting in the inhibition of IFN-alpha/beta- and IFN-gamma-mediated transcriptional activation. Finally, the M2 interaction with the p32 protein appeared to contribute to its ability to inhibit IFN-mediated transcriptional activation. These results indicate that gammaHV68 harbors a latency-associated M2 gene that antagonizes IFN-mediated host innate immunity and thus could play an important role in the establishment and maintenance of viral latency in infected animals.
Journal of Virology 12/2004; 78(22):12416-27. · 5.40 Impact Factor
-
Robert E Means
[show abstract]
[hide abstract]
ABSTRACT: Herpesvirus saimiri (HVS) is a gamma(2)-herpesvirus sharing genomic colinearity and a high degree of functional homology with HHV-8. To begin exploring the correlates of HVS infectivity and neutralization, we designed and implemented a new reporter assay. Using this assay, we could demonstrate that HVS neutralizing antibodies are present at high levels in naturally infected squirrel monkeys and are strongly induced after pathogenic, experimental infection of common marmosets. Further, we demonstrated that viral entry is influenced by cellular glycosaminoglycans and that, similar to HHV-8, soluble heparin is capable of blocking infectivity. We next cloned and characterized the positional homologue of HHV-8 K8.1, HVS Orf51. N-glycosidase F treatment indicates that like K8.1, Orf51 is a glycoprotein. Found in the viral particle, it localizes to the endoplasmic reticulum of expressing cells. Like K8.1, Orf51 could bind to agarose-conjugated heparin, implicating this molecule in viral attachment to cells. These studies provide the groundwork for additional experiments into the role that this protein may be playing in viral pathogenicity, persistence, and cell tropism.
Virology 09/2004; 326(1):67-78. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Through co-evolution with their hosts, viruses have developed a variety of immune escape and control mechanisms. In addition to strategies used to avoid the cellular and humoral immune responses, many viral families encode proteins capable of neutralizing the host's first line of defense, complement. The diversity of these complement avoidance mechanisms and proposed mechanisms by which viruses not only avoid, but also use the immune system to their advantage are discussed.
Trends in Microbiology 11/2003; 11(10):449-52. · 7.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Most rhesus macaques infected with simian immunodeficiency virus SIVmac239 with nef deleted (either Delta nef or Delta nef Delta vpr Delta US [Delta 3]) control viral replication and do not progress to AIDS. Some monkeys, however, develop moderate viral load set points and progress to AIDS. When simian immunodeficiency viruses (SIVs) recovered from two such animals (one Delta nef and the other Delta 3) were serially passaged in rhesus monkeys, the SIVs derived from both lineages were found to consistently induce moderate viral loads and disease progression. Analysis of viral sequences in the serially passaged derivatives revealed interesting changes in three regions: (i) an unusually high number of predicted amino acid changes (12 to 14) in the cytoplasmic domain of gp41, most of which were in regions that are usually conserved; these changes were observed in both lineages; (ii) an extreme shortening of nef sequences in the region of overlap with U3; these changes were observed in both lineages; and (iii) duplication of the NF-kappa B binding site in one lineage only. Neither the polymorphic gp41 changes alone nor the U3 deletion alone appeared to be responsible for increased replicative capacity because recombinant SIVmac239 Delta nef, engineered to contain either of these changes, induced moderate viral loads in only one of six monkeys. However, five of six monkeys infected with recombinant SIVmac239 Delta nef containing both TM and U3 changes did develop persisting moderate viral loads. These genetic changes did not increase lymphoid cell-activating properties in the monkey interleukin-2-dependent T-cell line 221, but the gp41 changes did increase the fusogenic activity of the SIV envelope two- to threefold. These results delineate sequence changes in SIV that can compensate for the loss of the nef gene to partially restore replicative and pathogenic potential in rhesus monkeys.
Journal of Virology 07/2003; 77(12):6823-35. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Herpesvirus saimiri Tip associates with Lck and downregulates Lck signal transduction. Here we demonstrate that Tip targets a lysosomal protein p80, which consists of an N-terminal WD repeat domain and a C-terminal coiled-coil domain. Interaction of Tip with p80 facilitated lysosomal vesicle formation and subsequent recruitment of Lck into the lysosomes for degradation. Consequently, Tip interactions with Lck and p80 result in downregulation of T cell receptor (TCR) and CD4 surface expression. Remarkably, these actions of Tip are functionally and genetically separable: the N-terminal p80 interaction is responsible for TCR downregulation and the C-terminal Lck interaction is responsible for CD4 downregulation. Thus, lymphotropic herpesvirus has evolved an elaborate mechanism to deregulate lymphocyte receptor expression to disarm host immune control.
Immunity 09/2002; 17(2):221-33. · 21.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Kaposi's sarcoma is an inflammatory cytokine-mediated angioproliferative disease which is triggered by infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV contains an open reading frame, K14, that has significant homology with cellular OX2, designated viral OX2 (vOX2). In this report, we demonstrate that vOX2 encodes a glycosylated cell surface protein with an apparent molecular mass of 55 kDa. Purified glycosylated vOX2 protein dramatically stimulated primary monocytes, macrophages, and dendritic cells to produce the inflammatory cytokines interleukin 1beta (IL-1beta), IL-6, monocyte chemoattractant protein 1, and TNF-alpha. Furthermore, expression of vOX2 on B lymphocytes stimulated monocytes to produce inflammatory cytokines in mixed culture. These results demonstrate that like its cellular counterpart, vOX2 targets myeloid-lineage cells, but unlike cellular OX2, which delivers a restrictive signal, KSHV vOX2 provides an activating signal, resulting in the production of inflammatory cytokines. Thus, this is a novel viral strategy where KSHV has acquired the cellular OX2 gene to induce inflammatory cytokine production, which potentially promotes the cytokine-mediated angiogenic proliferation of KSHV-infected cells.
Journal of Virology 06/2002; 76(10):4688-98. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The battle between viruses and their hosts is beautiful in its complexity. The interplay between viral proteins and the immune system has taught researchers much about not just the virus, but also the molecular mechanisms underlying the immune response. With additional evasion strategies constantly being described, this avenue of research is still rich with potential discoveries. In this review, we examine a number of proteins encoded by Kaposi's sarcoma herpesvirus (HHV-8) and detail how they aid the virus in escape from immune system elimination. We include, where possible, examples from other homologous viral systems.
Frontiers in Bioscience 06/2002; 7:e185-203. · 3.52 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
The EMBO Journal 05/2002; 21(7):1638-49. · 9.20 Impact Factor
