Membrane-associated and secreted forms of the Rhesus macaque rhadinovirus-encoded CD200 homologue and cellular CD200 demonstrate differential effects on Rhesus Macaque CD200 Receptor signaling and regulation of myeloid cell activation.

The CD200-CD200R pathway is involved in inhibition of immune responses, and the importance of this pathway to infectious disease is highlighted by the fact that viral CD200 (vCD200) molecules have been found to be encoded by several DNA viruses, including the human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), and the closely related rhesus macaque rhadinovirus (RRV). KSHV vCD200 is the most extensively studied vCD200 molecule, however, the only herpesvirus vCD200 molecule to be examined in vivo is that encoded by RRV. Our prior studies have demonstrated that RRV vCD200 is a functional CD200 homologue that is capable of affecting immune responses in vivo, and further, that RRV can express a secreted form of vCD200 (vCD200-Sec) during infection. Despite this information, RRV vCD200 has not been examined specifically for effects on RM CD200R signaling, and the functionality of vCD200-Sec has not been examined in any context. Thus, we developed an in vitro model system in which B cells expressing vCD200 were utilized to assess the effects of this molecule on the regulation of myeloid cells expressing RM CD200R, mimicking interactions that are predicted to occur in vivo Our findings suggest that RRV vCD200 can bind and induce functional signals through RM CD200R, while vCD200-Sec represents a non-functional protein incapable of affecting CD200R signaling. We also provide the first demonstration of the function of RM CD200, which appears to possess more robust signaling capabilities than RRV vCD200, and also show that KSHV vCD200 does not efficiently induce signaling via RM CD200R.IMPORTANCE Viral CD200 homologues are encoded by KSHV and the closely related RRV. Though RRV vCD200 has been examined, questions still exist in regard to the ability of this molecule to induce signaling via rhesus macaque CD200R, as well as the potential function of a secreted form of vCD200. Further, all previous in vitro studies of RRV vCD200 have utilized an Fc fusion protein to examine functionality, which does not replicate the structural properties of the membrane-associated form of vCD200 that is naturally produced during RRV infection. In this study, we demonstrate for the first time that membrane-expressed RRV vCD200 is capable of inducing signal transduction via RM CD200R, while the secreted form of vCD200 appears to be non-functional. Further, we also demonstrate that RM CD200 induces signaling via RM CD200R, and is more robust than RRV vCD200, while KSHV vCD200 does not appear to induce efficient signaling via RM CD200R.

Molecular analysis of lymphoid tissue from rhesus macaque rhadinovirus-infected monkeys identifies alterations in host genes associated with oncogenesis.

Rhesus macaque (RM) rhadinovirus (RRV) is a simian gamma-2 herpesvirus closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV). RRV is associated with the development of diseases in simian immunodeficiency virus (SIV) co-infected RM that resemble KSHV-associated pathologies observed in HIV-infected humans, including B cell lymphoproliferative disorders (LPD) and lymphoma. Importantly, how de novo KSHV infection affects the expression of host genes in humans, and how these alterations in gene expression affect viral replication, latency, and disease is unknown. The utility of the RRV/RM infection model provides a novel approach to address these questions in vivo, and utilizing the RRV bacterial artificial chromosome (BAC) system, the effects of specific viral genes on host gene expression patterns can also be explored. To gain insight into the effects of RRV infection on global host gene expression patterns in vivo, and to simultaneously assess the contributions of the immune inhibitory viral CD200 (vCD200) molecule to host gene regulation, RNA-seq was performed on pre- and post-infection lymph node (LN) biopsy samples from RM infected with either BAC-derived WT (n = 4) or vCD200 mutant RRV (n = 4). A variety of genes were identified as being altered in LN tissue samples due to RRV infection, including cancer-associated genes activation-induced cytidine deaminase (AICDA), glypican-1 (GPC1), CX3C chemokine receptor 1 (CX3CR1), and Ras dexamethasone-induced 1 (RasD1). Further analyses also indicate that GPC1 may be associated with lymphomagenesis. Finally, comparison of infection groups identified the differential expression of host gene thioredoxin interacting protein (TXNIP), suggesting a possible mechanism by which vCD200 negatively affects RRV viral loads in vivo.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti-IL-15- and control-treated groups in both cohorts. However, RM treated with anti-IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling.

Interferon (IFN) production and the subsequent induction of IFN-stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are promyelocytic leukemia nuclear bodies (PML-NBs), which are subnuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologs of IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN, while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3; however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our present study, we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription.IMPORTANCE KSHV and RRV encode a unique set of homologs of cellular IFN regulatory factors, termed vIRFs, which are hypothesized to help these viruses evade the innate immune response and establish infections in their respective hosts. Our work elucidates the role of one RRV vIRF, R12, and demonstrates that RRV can dampen the type I IFN response downstream of IFN signaling, which would be important for establishing a successful infection in vivo.

Gammaherpesvirus infection and malignant disease in rhesus macaques experimentally infected with SIV or SHIV.

Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.

Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.

UNLABELLED: Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3' untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. IMPORTANCE: Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.

Immunopathology of Japanese macaque encephalomyelitis is similar to multiple sclerosis.

Japanese macaque encephalomyelitis (JME) is an inflammatory demyelinating disease that occurs spontaneously in a colony of Japanese macaques (JM) at the Oregon National Primate Research Center. Animals with JME display clinical signs resembling multiple sclerosis (MS), and magnetic resonance imaging reveals multiple T2-weighted hyperintensities and gadolinium-enhancing lesions in the central nervous system (CNS). Here we undertook studies to determine if JME possesses features of an immune-mediated disease in the CNS. Comparable to MS, the CNS of animals with JME contain active lesions positive for IL-17, CD4+ T cells with Th1 and Th17 phenotypes, CD8+ T cells, and positive CSF findings.

A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response.

UNLABELLED: The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-ß) transcription, fails to effectively bind to the IFN-ß promoter, thus inhibiting the activation of IFN-ß genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-ß promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.

The rhesus rhadinovirus CD200 homologue affects immune responses and viral loads during in vivo infection.

UNLABELLED: Rhesus macaque rhadinovirus (RRV) is a gammaherpesvirus of rhesus macaque (RM) monkeys that is closely related to human herpesvirus 8 (HHV-8)/Kaposi's Sarcoma-associated herpesvirus (KSHV), and it is capable of inducing diseases in simian immunodeficiency virus (SIV)-infected RM that are similar to those seen in humans coinfected with HIV and HHV-8. Both HHV-8 and RRV encode viral CD200 (vCD200) molecules that are homologues of cellular CD200, a membrane glycoprotein that regulates immune responses and helps maintain immune homeostasis via interactions with the CD200 receptor (CD200R). Though the functions of RRV and HHV-8 vCD200 molecules have been examined in vitro, the precise roles that these viral proteins play during in vivo infection remain unknown. Thus, to address the contributions of RRV vCD200 to immune regulation and disease in vivo, we generated a form of RRV that lacked expression of vCD200 for use in infection studies in RM. Our data indicated that RRV vCD200 expression limits immune responses against RRV at early times postinfection and also impacts viral loads, but it does not appear to have significant effects on disease development. Further, examination of the distribution pattern of CD200R in RM indicated that this receptor is expressed on a majority of cells in peripheral blood mononuclear cells, including B and T cells, suggesting potentially wider regulatory capabilities for both vCD200 and CD200 that are not strictly limited to myeloid lineage cells. In addition, we also demonstrate that RRV infection affects CD200R expression levels in vivo, although vCD200 expression does not play a role in this phenomenon. IMPORTANCE: Cellular CD200 and its receptor, CD200R, compose a pathway that is important in regulating immune responses and is known to play a role in a variety of human diseases. A number of pathogens have been found to modulate the CD200-CD200R pathway during infection, including human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma and B cell neoplasms in AIDS patients, and a closely related primate virus, rhesus macaque rhadinovirus (RRV), which infects and induces disease in rhesus macaque monkeys. HHV-8 and RRV encode homologues of CD200, termed vCD200, which are thought to play a role in preventing immune responses against these viruses. However, neither molecule has been studied in an in vivo model of infection to address their actual contributions to immunoregulation and disease. Here we report findings from our studies in which we analyzed the properties of a mutant form of RRV that lacks vCD200 expression in infected rhesus macaques.

Rhesus macaque rhadinovirus-associated disease.

Rhesus macaque rhadinovirus (RRV) is a gamma-2 herpesvirus that naturally infects rhesus macaque (RM) monkeys and is closely related to human herpesvirus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). Infection of immunodeficient RM induces disease in infected RM that resembles KSHV-associated pathologies. Importantly, RRV possesses homologues of KSHV ORFs that are postulated to play a role in disease development. As such, RRV has emerged as a prominent in vivo model system for examining mechanisms of infection and disease of these pathogenic herpesviruses, and has provided unique insight into how these viruses cause disease.

Viral interferon regulatory factors are critical for delay of the host immune response against rhesus macaque rhadinovirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host.

Viral interferon regulatory factors decrease the induction of type I and type II interferon during rhesus macaque rhadinovirus infection.

Kaposi's sarcoma-associated herpesvirus and rhesus macaque rhadinovirus (RRV), two closely related gammaherpesviruses, are unique in their expression of viral homologs of cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). To assess the role of vIRFs during de novo infection, we have utilized the bacterial artificial chromosome clone of wild-type RRV(17577) (WT(BAC) RRV) to generate a recombinant virus with all 8 of the vIRFs deleted (vIRF-ko RRV). The infection of primary rhesus fibroblasts and peripheral blood mononuclear cells (PBMCs) with vIRF-ko RRV resulted in earlier and increased induction of type I interferon (IFN) (IFN-α/ß) and type II IFN (IFN-γ). Additionally, plasmacytoid dendritic cells maintained higher levels of IFN-α production in PBMC cultures infected with vIRF-ko RRV than in cultures infected with WT(BAC) RRV. Moreover, the nuclear accumulation of phosphorylated IRF-3, which is necessary for the induction of type I IFN, was also inhibited following WT(BAC) RRV infection. These findings demonstrate that during de novo RRV infection, vIRFs are inhibiting the induction of IFN at the transcriptional level, and one potential mechanism for this is the disruption of the activation and localization of IRF-3.

Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques.

Rhesus rhadinovirus (RRV), a primate gamma-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3' to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered >600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.

Prevalence of viremia and oral shedding of rhesus rhadinovirus and retroperitoneal fibromatosis herpesvirus in large age-structured breeding groups of rhesus macaques (Macaca mulatta).

We performed a cross-sectional study to estimate the prevalence of 2 gamma-2-herpesviruses, rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), in breeding colonies of rhesus macaques. Of 90 animals selected for sampling, 73 (81%) were positive for RRV, which was detected only in blood in 22 (24%), only in saliva in 15 (16%), and in both blood and saliva in 36 (40%). Detection of RRV DNA in blood and saliva was significantly higher in animals younger than 2 y. In comparison, RFHV was detected in 40 (44%) of the 90 animals: only in blood in 5 (6%), only in saliva in 26 (29%), and in both blood and saliva in 9 (10%). Dual infection was detected in 38 (42%) animals; RFHV was only detected in coinfections. The mean RRV genome copy number in blood was significantly higher than that for RFHV. Age was a significant predictor of RRV copy number in blood and RFHV copy number in saliva. Of the 90 animals, 88 (98%) were positive for rhadinoviral antibodies on an immunofluorescent assay. Both RRV and RFHV are highly endemic in socially housed breeding colonies of rhesus macaques, and their patterns of infection are similar to that for the betaherpesvirus rhesus cytomegalovirus.

Characterization of the complement inhibitory function of rhesus rhadinovirus complement control protein (RCP).

Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.

Rhesus macaque rhadinovirus-associated non-Hodgkin lymphoma: animal model for KSHV-associated malignancies.

Rhesus macaque rhadinovirus (RRV) is closely related to Kaposi sarcoma-associated herpesvirus (KSHV) and is associated with the development of B-cell hyperplasia and persistent lymphadenopathy resembling multicentric Castleman disease in rhesus macaques (RMs) coinfected with simian immunodeficiency virus (SIV). Here we investigated whether RMs experimentally infected with SIV and RRV can develop other disease manifestations observed in HIV- and KSHV-infected patients. As reported earlier, inoculation of SIV-infected RMs with RRV results in persistent RRV infection, whereas immunocompetent animals infected with RRV exhibit viremia 2 weeks after infection, followed by a period of no virus detection until they are subsequently made immunodeficient by SIV infection. A subset of animals developed abnormal cellular proliferations characterized as extranodal lymphoma and a proliferative mesenchymal lesion. In situ hybridization and immunohistochemistry analysis indicate RRV is present in both malignancies, and DNA microarray analysis detected viral interleukin-6 (vIL-6) and viral FLICE-like inhibitory protein (vFLIP) transcripts. Reverse-transcriptase polymerase chain reaction analysis confirmed vIL-6 and vFLIP expression, and that of RRV open reading frames 72 and 73, homologs of KSHV open reading frames shown to be expressed in primary effusion lymphoma. These data support the utility of the RRV-/SIV-infected RM as an excellent animal model to investigate KSHV-like pathogenesis.

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Construction of an infectious rhesus rhadinovirus bacterial artificial chromosome for the analysis of Kaposi's sarcoma-associated herpesvirus-related disease development.

Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV(17577)) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV(17577), producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.

Rhesus rhadinovirus R15 encodes a functional homologue of human CD200.

A viral CD200 homologue (vCD200) encoded by open reading frame R15 of rhesus rhadinovirus (RRV), a gammaherpesvirus closely related to human herpesvirus 8 (HHV-8), is described here. RRV vCD200 shares 30% and 28% amino acid identity with human CD200 (huCD200) and HHV-8 vCD200, respectively. In vitro analysis indicated that an Fc fusion (vCD200-Fc) is expressed as a glycoprotein with a core molecular mass of 53 kDa. Utilizing monoclonal antibodies raised against vCD200-Fc, vCD200 expression was detected on the surfaces of and within supernatants from infected fibroblasts. Furthermore, in vitro assays demonstrated that vCD200-Fc treatment of monocyte-derived macrophages reduces tumor necrosis factor transcript and protein levels, implying that RRV encodes a functional vCD200.

Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIV(KU-1bMC33)) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques.

Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5' to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV(Vpenv)) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV(Vpenv)-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV(Vpenv)-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) and monitored for 6 months for CD4(+) T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIV(Vpenv) caused a severe CD4(+) T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4(+) T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV(Vpenv) with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.