Rhesus Rhadinovirus Research Articles

Characterization of two divergent lineages of macaque rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus.

We have cloned and characterized the entire DNA polymerase gene and flanking regions from Kaposi's sarcoma-associated herpesvirus (KSHV) and two closely related macaque homologs of KSHV, retroperitoneal fibromatosis-associated herpesvirus-Macaca nemestrina (RFHVMn) and -Macaca mulatta (RFHVMm). We have also identified and partially characterized the corresponding genomic region of another KSHV-like herpesvirus, provisionally named "M. nemestrina rhadinovirus type 2 (MneRV-2)," with close similarity to rhesus rhadinovirus (RRV). A sequence comparison of these four macaque viruses and two KSHV-like gammaherpesviruses recently identified in African green monkeys, Chlorocebus rhadinovirus types 1 and 2 (ChRV-1 and ChRV-2) reveals the presence of two distinct lineages of KSHV-like rhadinoviruses in Old World primates. The first rhadinovirus lineage consists of KSHV and its closely related homologs RFHVMn, RFHVMm, and ChRV-1, while the second more distantly related lineage consists of RRV, MneRV-2, and ChRV-2. Our findings raise the possibility of the existence of another human KSHV-like herpesvirus belonging to the second rhadinovirus lineage.

The primary sequence of rhesus monkey rhadinovirus isolate 26-95: sequence similarities to Kaposi's sarcoma-associated herpesvirus and rhesus monkey rhadinovirus isolate 17577.

The primary sequence of the long unique region L-DNA (L for low GC) of rhesus monkey rhadinovirus (RRV) isolate 26-95 was determined. The L-DNA consists of 130,733 bp that contain 84 open reading frames (ORFs). The overall organization of the RRV26-95 genome was found to be very similar to that of human Kaposi sarcoma-associated herpesvirus (KSHV). BLAST search analysis revealed that in almost all cases RRV26-95 coding sequences have a greater degree of similarity to corresponding KSHV sequences than to other herpesviruses. All of the ORFs present in KSHV have at least one homologue in RRV26-95 except K3 and K5 (bovine herpesvirus-4 immediate-early protein homologues), K7 (nut-1), and K12 (Kaposin). RRV26-95 contains one MIP-1 and eight interferon regulatory factor (vIRF) homologues compared to three MIP-1 and four vIRF homologues in KSHV. All homologues are correspondingly located in KSHV and RRV with the exception of dihydrofolate reductase (DHFR). DHFR is correspondingly located near the left end of the genome in RRV26-95 and herpesvirus saimiri (HVS), but in KSHV the DHFR gene is displaced 16,069 nucleotides in a rightward direction in the genome. DHFR is also unusual in that the RRV26-95 DHFR more closely resembles HVS DHFR (74% similarity) than KSHV DHFR (55% similarity). Of the 84 ORFs in RRV26-95, 83 contain sequences similar to the recently determined sequences of the independent RRV isolate 17577. RRV26-95 and RRV17577 sequences differ in that ORF 67.5 sequences contained in RRV26-95 were not found in RRV17577. In addition, ORF 4 is significantly shorter in RRV26-95 than was reported for RRV17577 (395 versus 645 amino acids). Only four of the corresponding ORFs between RRV26-95 and RRV17577 exhibited less than 95% sequence identity: glycoproteins H and L, uracil DNA glucosidase, and a tegument protein (ORF 67). Both RRV26-95 and RRV17577 have unique ORFs between positions 21444 to 21752 and 110910 to 114899 in a rightward direction and from positions 116524 to 111082 in a leftward direction that are not found in KSHV. Our analysis indicates that RRV26-95 and RRV17577 are clearly independent isolates of the same virus species and that both are closely related in structural organization and overall sequence to KSHV. The availability of detailed sequence information, the ability to grow RRV lytically in cell culture, and the ability to infect monkeys experimentally with RRV will facilitate the construction of mutant strains of virus for evaluating the contribution of individual genes to biological properties.

Activation of lymphocyte signaling by the R1 protein of rhesus monkey rhadinovirus.

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the human Kaposi's sarcoma-associated herpesvirus (KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.

Signaling activities of gammaherpesvirus membrane proteins.

Members of the Herpesviridae family have large doublestranded DNA genomes and replicate in the nucleus of the host cell. Based on genomic organization and biological characteristics, herpesviruses are classified into three subfamilies: alpha, beta, and gamma (Fig. 1A). The gammaherpesviruses replicate and persist in lymphoid cells, but some are capable of undergoing lytic replication in epithelial or fibroblast cells. These viruses can be subdivided into two genera: lymphocryptoviruses (gamma-1) and rhadinoviruses (gamma-2) (Fig. 1A). The lymphocryptoviruses (gamma-1) include Epstein-Barr virus (EBV) or Human herpesvirus 4, Lymphocryptovirus of rhesus monkeys, and Herpesvirus papio of baboons, whereas the rhadinoviruses (gamma-2) include Herpesvirus saimiri (HVS), Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human herpesvirus 8, Rhesus monkey rhadinovirus (RRV), Equine herpesvirus 2, and Mouse herpesvirus 68 (Fig. 1B). The gammaherpesviruses, including HVS, EBV, KSHV, and RRV, are capable of establishing latent infection in lymphocytes. Both HVS and EBV have also been shown to transform lymphoid cells and to induce lymphoproliferative diseases in the natural or experimental host. EBV has been shown to be associated with several diseases in humans (16, 40, 78). These include infectious mononucleosis (IM), Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s disease (HD), and T-cell lymphomas (1, 11, 65, 83, 84, 95, 96, 108, 116, 141). Primary EBV infection is usually asymptomatic, but a proportion of EBV-infected individuals develop IM, a disease characterized by lymphadenopathy and fatigue, later in life. A rare disease called fatal IM or X-linked lymphoproliferative (XLP) syndrome is an EBV-dependent malignancy characterized by uncontrolled immunoblastic lymphomas which are consistently EBV positive (115). The genetic defect in XLP is in the SLAM-associated protein, SAP. The mutated SAP protein in XLP patients affects T/B-cell interactions induced by SLAM, leading to an inability to control the B-cell proliferation caused by EBV infections (123, 133). BL is a malignancy that principally affects children, especially those that live in regions of Africa with a high incidence of malaria (16). The tumor cells of these lymphomas are closely associated with EBV, with 100% of the lymphomas scoring positive for EBV. In other areas of the world, however, the occurrence of BL is more sporadic and a lower percentage of these lymphomas are EBV positive (65). BL is characterized by distinct chromosomal translocations of the c-myc oncogene and immunoglobulin promoter sequences, resulting in the deregulation of c-myc expression (23). Another EBV-associated disease is NPC, a malignancy of the squamous epithelium situated in the nasopharynx (117). EBV is consistently present in cases of epithelial dysplasia, and it is thought to have arisen from clonal expansion of latently infected cells (113, 116). The incidence of NPC is high in Southern China, Northern Africa, and Eskimo populations. HD is the most common malignancy in the Western world, with about 30 to 90% of all HD lymphomas being EBV positive. Like NPC, the EBV genomes in these tumor cells are monoclonal (139). EBV is also present in about 70% of all immunoblastic lymphomas in human immunodeficiency virus (HIV)-infected individuals and in 100% of immunoblastic lymphomas of immune-suppressed posttransplant patients. Recently, EBV, a primarily B-cell-tropic virus, has been detected in different types of human T-cell lymphomas. About 100% of all nasal T-cell lymphomas in Southeast Asia and 100% of T-cell tumors in XLP males contain EBV (65).

Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2 herpesvirus lineage among old world primates?

Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens.

A search for human herpesvirus 8(HHV-8) in HIV-1 infected mothers and their infants does not suggest vertical transmission of HHV-8

A search for human herpesvirus 8(HHV-8) in HIV-1 infected mothers and their infants does not suggest vertical transmission of HHV-8

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses.

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.

Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.

Rhesus rhadinovirus establishes a latent infection in B lymphocytes in vivo.

Recent DNA sequence analysis indicates that rhesus rhadinovirus (RRV) is a member of the lymphotropic gamma-2 herpesvirus family. To determine if RRV is lymphotropic, peripheral blood mononuclear cells from naturally infected monkeys were separated by immunomagnetic bead depletion and analyzed for the presence of RRV by virus isolation and nested PCR. The recovery and consistent detection of RRV in the CD20(+)-enriched fraction clearly demonstrates that B lymphocytes are a major site of virus persistence.

A rhesus macaque rhadinovirus related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 encodes a functional homologue of interleukin-6.

The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi's sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6). To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathione S-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays. Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner. Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6. Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells. These results demonstrate that RRV encodes an accessory protein with IL-6-like activity.

Identification of a thymidylate synthase gene within the genome of Chilo iridescent virus.

The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.

A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus.

A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus.