Dataset Information

Complete genome sequence of Pig-tailed macaque rhadinovirus 2 and its evolutionary relationship with rhesus macaque rhadinovirus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

ABSTRACT: Two rhadinovirus lineages have been identified in Old World primates. The rhadinovirus 1 (RV1) lineage consists of human herpesvirus 8, Kaposi's sarcoma-associated herpesvirus (KSHV), and closely related rhadinoviruses of chimpanzees, gorillas, macaques and other Old World primates. The RV2 rhadinovirus lineage is distinct and consists of closely related viruses from the same Old World primate species. Rhesus macaque rhadinovirus (RRV) is the RV2 prototype, and two RRV isolates, 26-95 and 17577, were sequenced. We determined that the pig-tailed macaque RV2 rhadinovirus, MneRV2, is highly associated with lymphomas in macaques with simian AIDS. To further study the role of rhadinoviruses in the development of lymphoma, we sequenced the complete genome of MneRV2 and identified 87 protein coding genes and 17 candidate microRNAs (miRNAs). A strong genome colinearity and sequence homology were observed between MneRV2 and RRV26-95, although the open reading frame (ORF) encoding the KSHV ORFK15 homolog was disrupted in RRV26-95. Comparison with MneRV2 revealed several genomic anomalies in RRV17577 that were not present in other rhadinovirus genomes, including an N-terminal duplication in ORF4 and a recombinative exchange of more distantly related homologs of the ORF22/ORF47 interacting glycoprotein genes. The comparison with MneRV2 has revealed novel genes and important conservation of protein coding domains and transcription initiation, termination, and splicing signals, which have added to our knowledge of RV2 rhadinovirus genetics. Further comparisons with KSHV and other RV1 rhadinoviruses will provide important avenues for dissecting the biology, evolution, and pathology of these closely related tumor-inducing viruses in humans and other Old World primates.This work provides the sequence characterization of MneRV2, the pig-tailed macaque homolog of rhesus rhadinovirus (RRV). MneRV2 and RRV belong to the rhadinovirus 2 (RV2) rhadinovirus lineage of Old World primates and are distinct but related to Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma. Pig-tailed macaques provide important models of human disease, and our previous studies have indicated that MneRV2 plays a causal role in AIDS-related lymphomas in macaques. Delineation of the MneRV2 sequence has allowed a detailed characterization of the genome structure, and evolutionary comparisons with RRV and KSHV have identified conserved promoters, splice junctions, and novel genes. This comparison provides insight into RV2 rhadinovirus biology and sets the groundwork for more intensive next-generation (Next-Gen) transcript and genetic analysis of this class of tumor-inducing herpesvirus. This study supports the use of MneRV2 in pig-tailed macaques as an important model for studying rhadinovirus biology, transmission and pathology.

SUBMITTER: Bruce AG

PROVIDER: S-EPMC4403432 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Complete genome sequence of Pig-tailed macaque rhadinovirus 2 and its evolutionary relationship with rhesus macaque rhadinovirus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

Bruce A Gregory AG Thouless Margaret E ME Haines Anthony S AS Pallen Mark J MJ Grundhoff Adam A Rose Timothy M TM

Journal of virology 20150121 7

<h4>Unlabelled</h4>Two rhadinovirus lineages have been identified in Old World primates. The rhadinovirus 1 (RV1) lineage consists of human herpesvirus 8, Kaposi's sarcoma-associated herpesvirus (KSHV), and closely related rhadinoviruses of chimpanzees, gorillas, macaques and other Old World primates. The RV2 rhadinovirus lineage is distinct and consists of closely related viruses from the same Old World primate species. Rhesus macaque rhadinovirus (RRV) is the RV2 prototype, and two RRV isolate ...[more]

PMID: 25609822

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Project description: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.

Project description:KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis- and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related γ2-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells.IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis- and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi's sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.

Project description: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.

Project description:The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens and may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action is well understood. We report here that in some cells but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

Dataset Information

Complete genome sequence of Pig-tailed macaque rhadinovirus 2 and its evolutionary relationship with rhesus macaque rhadinovirus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

Publications

Complete genome sequence of Pig-tailed macaque rhadinovirus 2 and its evolutionary relationship with rhesus macaque rhadinovirus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

Similar Datasets

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