Rhesus Rhadinovirus Research Articles

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

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-β) transcription, fails to effectively bind to the IFN-β promoter, thus inhibiting the activation of IFN-β 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-β 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. 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.

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.

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.

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

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. 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.

Maturation and vesicle-mediated egress of primate gammaherpesvirus rhesus monkey rhadinovirus require inner tegument protein ORF52.

The tegument layer of herpesviruses comprises a collection of proteins that is unique to each viral species. In rhesus monkey rhadinovirus (RRV), a close relative of the human oncogenic pathogen Kaposi's sarcoma-associated herpesvirus, ORF52 is a highly abundant tegument protein tightly associated with the capsid. We now report that ORF52 knockdown during RRV infection of rhesus fibroblasts led to a greater than 300-fold reduction in the viral titer by 48 h but had little effect on the number of released particles and caused only modest reductions in the levels of intracellular viral genomic DNA and no appreciable change in viral DNA packaging into capsids. These data suggested that the lack of ORF52 resulted in the production and release of defective particles. In support of this interpretation, transmission electron microscopy (TEM) revealed that without ORF52, capsid-like particles accumulated in the cytoplasm and were unable to enter egress vesicles, where final tegumentation and envelopment normally occur. TEM also demonstrated defective particles in the medium that closely resembled the accumulating intracellular particles, having neither a full tegument nor an envelope. The disruption in tegument formation from ORF52 suppression, therefore, prevented the incorporation of ORF45, restricting its subcellular localization to the nucleus and appearing, by confocal microscopy, to inhibit particle transport toward the periphery. Ectopic expression of small interfering RNA (siRNA)-resistant ORF52 was able to partially rescue all of these phenotypic changes. In sum, our results indicate that efficient egress of maturing virions and, in agreement with studies on murine gammaherpesvirus 68 (MHV-68), complete tegumentation and secondary envelopment are dependent on intact ORF52. The tegument, or middle layer, of herpesviruses comprises both viral and cellular proteins that play key roles in the viral life cycle. A subset of these proteins is present only within members of one of the three subfamilies (alphaherpesviruses, betaherpesviruses, or gammaherpesviruses) of Herpesviridae. In this report, we show that the gammaherpesvirus-specific tegument protein ORF52 is critical for maturation of RRV, the closest relative of Kaposi's sarcoma-associated herpesvirus (KSHV) (a human cancer-causing pathogen) that has undergone this type of analysis. Without ORF52, the nascent subviral particles are essentially stuck in maturation limbo, unable to acquire the tegument or outer (envelope) layers. This greatly attenuates infectivity. Our data, together with earlier work on a murine homolog, as well as a more distantly related human homolog, provide a more complete understanding of how early protein interactions involving virus-encoded tegument proteins are critical for virus assembly and are also, therefore, potentially attractive therapeutic targets.

Progressive accumulation of activated ERK2 within highly stable ORF45-containing nuclear complexes promotes lytic gammaherpesvirus infection.

De novo infection with the gammaherpesvirus Rhesus monkey rhadinovirus (RRV), a close homolog of the human oncogenic pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV), led to persistent activation of the MEK/ERK pathway and increasing nuclear accumulation of pERK2 complexed with the RRV protein, ORF45 (R45) and cellular RSK. We have previously shown that both lytic gene expression and virion production are dependent on the activation of ERK [1]. Using confocal microscopy, sequential pull-down assays and FRET analyses, we have demonstrated that pERK2-R45-RSK2 complexes were restricted to the nucleus but that the activated ERK retained its ability to phosphorylate nuclear substrates throughout infection. Furthermore, even with pharmacologic inhibition of MEK beginning at 48 h p.i., pERK2 but not pERK1, remained elevated for at least 10 h, showing first order decay and a half-life of nearly 3 hours. Transfection of rhesus fibroblasts with R45 alone also led to the accumulation of nuclear pERK2 and addition of exogenous RSK augmented this effect. However, knock down of RSK during bona fide RRV infection had little to no effect on pERK2 accumulation or virion production. The cytoplasmic pools of pERK showed no co-localization with either RSK or R45 but activation of pERK downstream targets in this compartment was evident throughout infection. Together, these observations suggest a model in which R45 interacts with pERK2 to promote its nuclear accumulation, thereby promoting lytic viral gene expression while also preserving persistent and robust activation of both nuclear and cytoplasmic ERK targets.

A critical Sp1 element in the rhesus rhadinovirus (RRV) Rta promoter confers high-level activity that correlates with cellular permissivity for viral replication

KSHV establishes characteristic latent infections in vitro, while RRV, a related macaque rhadinovirus, establishes characteristic permissive infections with virus replication. We identified cells that are not permissive for RRV replication and recapitulate the latent KSHV infection and reactivation processes. The RRV replication and transactivator (Rta) promoter was characterized in permissive and non-permissive cells and compared to the KSHV Rta promoter. Both promoters contained a critical Sp1 element, had equivalent activities in different cell types, and were inhibited by LANA. RRV and KSHV infections were non-permissive in cells with low Rta promoter activity. While RRV infections were permissive in cells with high basal promoter activity, KSHV infections remained non-permissive. Our studies suggest that RRV lacks the Rta-inducible LANA promoter that is responsible for LANA inhibition of the KSHV Rta promoter and induction of latency during KSHV infection. Instead, the outcome of RRV infection is determined by host factors, such as Sp1.

Rhesus Monkey Rhadinovirus Uses Eph Family Receptors for Entry into B Cells and Endothelial Cells but Not Fibroblasts

Cellular Ephrin receptor tyrosine kinases (Ephrin receptors, Ephs) were found to interact efficiently with the gH/gL glycoprotein complex of the rhesus monkey rhadinovirus (RRV). Since EphA2 was recently identified as a receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV) (Hahn et al., Nature Medicine 2012), we analyzed RRV and KSHV in parallel with respect to Eph-binding and Eph-dependent entry. Ten of the 14 Eph proteins, including both A- and B-type, interacted with RRV gH/gL. Two RRV strains with markedly different gH/gL sequences exhibited similar but slightly different binding patterns to Ephs. gH/gL of KSHV displayed high affinity towards EphA2 but substantially weaker binding to only a few other Ephs of the A-type. Productive entry of RRV 26-95 into B cells and into endothelial cells was essentially completely dependent upon Ephs since expression of a GFP reporter cassette from recombinant virus could be blocked to greater than 95% by soluble Eph decoys using these cells. In contrast, entry of RRV into fibroblasts and epithelial cells was independent of Ephs by these same criteria. Even high concentrations and mixtures of soluble Eph decoys were not able to reduce by any appreciable extent the number of fibroblasts and epithelial cells productively entered by RRV. Thus, RRV is similar to its close relative KSHV in the use of Eph family receptors for productive entry into B cells and endothelial cells. However, RRV uses a separate, distinct, Eph-independent pathway for productive entry into fibroblasts and epithelial cells. Whether KSHV also uses an Eph-independent pathway in some circumstances or to some extent remains to be determined.

Activity and Mechanism of Action of HDVD, a Novel Pyrimidine Nucleoside Derivative with High Levels of Selectivity and Potency against Gammaherpesviruses

A novel nucleoside analogue, 1-[(2S,4S-2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(1H,3H)-dione, or HDVD, was evaluated against a wide variety of herpesviruses and was found to be a highly selective inhibitor of replication of the gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). HDVD had also a pronounced inhibitory activity against murine herpesvirus 68 (MHV-68) and herpes simplex virus 1 (HSV-1). In contrast, replication of herpesvirus saimiri (HVS), HSV-2, and varicella-zoster virus (VZV) was weakly inhibited by the compound, and no antiviral activity was determined against human cytomegalovirus (HCMV) and rhesus rhadinovirus (RRV). The HDVD-resistant virus phenotype contained point mutations in the viral thymidine kinase (TK) of HSV-1, MHV-68, and HVS isolates. These mutations conferred cross-resistance to other TK-dependent drugs, with the exception of an MHV-68 mutant (E358D) that exhibited resistance only to HDVD. HSV-1 and HVS TK-mutants isolated under selective pressure with bromovinyldeoxyuridine (BVDU) also showed reduced sensitivity to HDVD. Oral treatment with HDVD and BVDU was assessed in an intranasal model of MHV-68 infection in BALB/c mice. In contrast to BVDU treatment, HDVD-treated animals showed a reduction in viral DNA loads and diminished viral gene expression during acute viral replication in the lungs in comparison to levels in untreated controls. The valyl ester prodrug of HDVD (USS-02-71-44) suppressed the latent infection in the spleen to a greater extent than HDVD. In the present study, HDVD emerged as a highly potent antiviral with a unique spectrum of activity against herpesviruses, in particular, gammaherpesviruses, and may be of interest in the treatment of virus-associated diseases.

Exploitation of Cellular Cytoskeletons and Signaling Pathways for Cell Entry by Kaposi’s Sarcoma-Associated Herpesvirus and the Closely Related Rhesus Rhadinovirus

As obligate intracellular pathogens, viruses depend on the host cell machinery to complete their life cycle. Kaposi’s sarcoma-associated herpes virus (KSHV) is an oncogenicvirus causally linked to the development of Kaposi’s sarcoma and several other lymphoproliferative malignancies. KSHV entry into cells is tightly regulated by diverse viral and cellular factors. In particular, KSHV actively engages cellular integrins and ubiquitination pathways for successful infection. Emerging evidence suggests that KSHV hijacks both actin and microtubule cytoskeletons at different phases during entry into cells. Here, we review recent findings on the early events during primary infection of KSHV and its closely related primate homolog rhesus rhadinovirus with highlights on the regulation of cellular cytoskeletons and signaling pathways that are important for this phase of virus life cycle.

Conservation of a Triple-Helix-Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

Distinct Roles for Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 in the Structure and Production of a Primate Gammaherpesvirus

During their progression from intranuclear capsids to mature trilaminar virions, herpesviruses incorporate an extensive array of viral as well as a smaller subset of cellular proteins. Our laboratory previously reported that rhesus monkey rhadinovirus (RRV), a close homolog of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is comprised of at least 33 different virally encoded proteins. In the current study, we found that RRV infection activated the extracellular signal-regulated kinase (ERK) pathway and nascent virions preferentially incorporated the activated form of ERK2 (pERK2) into the tegument. This was evident even in the face of greatly diminished stores of intracellular ERK2, suggesting a clear bias toward the incorporation of pERK2 into the RRV particle. Similar to earlier findings with KSHV, activation of ERK was essential for the production of lytic viral proteins and virions. Knockdown of intracellular ERK, however, failed to inhibit virus production, likely due to maintenance of residual pools of intracellular pERK2. Paradoxically, selective knockdown of ERK1 enhanced virion production nearly 5-fold and viral titers more than 10-fold. These data are the first to implicate ERK1 as a negative regulator of lytic replication in a herpesvirus and the first to demonstrate the incorporation of an activated signaling molecule within a herpesvirus. Together, the results further our understanding of how herpesviruses interact with host cells during infection and demonstrate how this family of viruses can exploit cellular signal transduction pathways to modulate their own replication.

Viral FLICE Inhibitory Protein of Rhesus Monkey Rhadinovirus Inhibits Apoptosis by Enhancing Autophagosome Formation

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to human herpesvirus 8 (HHV8). RRV encodes viral FLICE inhibitory protein (vFLIP), which has death effector domains. Little is known about RRV vFLIP. This study intended to examine its function in apoptosis. Here we found that RRV vFLIP inhibits apoptosis induced by tumor necrosis factor-α (TNF-α) and cycloheximide. In HeLa cells with vFLIP expression, the cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than those in controls. Cell viability of HeLa cells with vFLIP expression was significantly higher than control cells after apoptosis induction. However, RRV vFLIP appears unable to induce NF-κB signaling when tested in NF-κB reporter assay. RRV vFLIP was able to enhance cell survival under starved conditions or apoptosis induction. At early time points after apoptosis induction, autophagosome formation was enhanced and LC3-II level was elevated in cells with vFLIP and, when autophagy was blocked with chemical inhibitors, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis by enhancing autophagy to maintain cell survival. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These results indicate that vFLIP protects cells against apoptosis by enhancing autophagosome formation to extend cell survival. The finding of vFLIP’s inhibition of apoptosis via the autophagy pathway provides insights of vFLIP in RRV pathogenesis.

Viral FLICE inhibitory protein of Rhesus monkey rhadinovirus inhibits apoptosis by enhancing autophagosome formation

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to human herpesvirus 8 (HHV8). RRV encodes viral FLICE inhibitory protein (vFLIP), which has death effector domains. Little is known about RRV vFLIP. This study intended to examine its function in apoptosis. Here we found that RRV vFLIP inhibits apoptosis induced by tumor necrosis factor-α (TNF-α) and cycloheximide. In HeLa cells with vFLIP expression, the cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than those in controls. Cell viability of HeLa cells with vFLIP expression was significantly higher than control cells after apoptosis induction. However, RRV vFLIP appears unable to induce NF-κB signaling when tested using NF-κB reporter assay. RRV vFLIP was able to enhance cell survival under starved conditions or apoptosis induction. At early time points after apoptosis induction, autophagosome formation was enhanced and LC3-II level was elevated in cells with vFLIP and, when autophagy was blocked with chemical inhibitors, these cells underwent apoptosis. Full length of vFLIP is needed for the function against apoptosis as truncation variants of vFLIP were unable to block apoptosis induction. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis by enhancing autophagy to maintain cell survival. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These findings indicate that vFLIP protects cells against apoptosis by enhancing autophagosome formation to extend cell survival. The finding of vFLIP’s inhibition of apoptosis via the autophagy pathway provides insights of vFLIP in RRV pathogenesis.

Microtubule- and Dynein-Dependent Nuclear Trafficking of Rhesus Rhadinovirus in Rhesus Fibroblasts

We investigated the role of microtubules in rhesus rhadinovirus (RRV) nuclear trafficking in rhesus fibroblasts. Intact microtubules and microtubule dynamics are required for RRV trafficking to perinuclear regions. RRV trafficking was reduced by an inhibitor of the dynein motor and overexpression of dynamitin. Furthermore, RRV particles are colocalized with microtubules and dynein proteins. These results highlight the important roles of microtubules and dynein-dynactin complexes in the transport of RRV particles to nuclei during primary infection.

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs

Open reading frame 57 (ORF57) of gamma-2 herpesviruses is a key regulator of viral gene expression. It has been reported to enhance the expression of viral genes by transcriptional, posttranscriptional, or translational activation mechanisms. Previously we have shown that the expression of gH and gL of rhesus monkey rhadinovirus (RRV), a close relative of the human Kaposi's sarcoma-associated herpesvirus (KSHV), could be dramatically rescued by codon optimization as well as by ORF57 coexpression (J. P. Bilello, J. S. Morgan, and R. C. Desrosiers, J. Virol. 82:7231-7237, 2008). We show here that ORF57 coexpression and codon optimization had similar effects, except that the rescue of expression by codon optimization was temporally delayed relative to that of ORF57 coexpression. The transfection of gL mRNA directly into cells with or without ORF57 coexpression and with or without codon optimization recapitulated the effects of these modes of induction on transfected DNA. These findings suggested an important role for the enhancement of mRNA stability and/or the translation of mRNA for these very different modes of induced expression. This conclusion was confirmed by several different measures of gH and gL mRNA stability and accumulation with or without ORF57 coexpression and with or without codon optimization. Our results indicate that RRV gH and gL expression is severely limited by the stability of the mRNA and that ORF57 coexpression and codon optimization independently induce gH and gL expression principally by allowing accumulation and translation of these mRNAs.

Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi's sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman's disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.

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

Rhesus rhadinovirus (RRV), a primate γ-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.

Accumulation of oxidized proteins in Herpesvirus infected cells

Oxidative stress gives rise to an environment that can be highly damaging to proteins, lipids, and DNA. Previous studies indicate that Herpesvirus infections cause oxidative stress in cells and in tissues. The biological consequences of virus-induced oxidative stress have not been characterized. Studies from many groups indicate that proteins which have been damaged through oxidative imbalances are either degraded by the 20S proteasome in a ubiquitin-independent fashion or form aggregates that are resistant to proteolysis. We have previously shown that herpes simplex virus type 1 (HSV-1) replication was significantly enhanced in the presence of the cellular antioxidant chaperone Hsp27, indicating a possible role for this protein in managing virus-induced oxidative stress. Here we show that oxidized proteins accumulate during infections with two distantly related herpesviruses, HSV-1 and Rhesus Rhadinovirus (RRV), a close relative of the Kaposi's sarcoma-associated herpesvirus (KSHV). The presence of oxidized proteins was not entirely unexpected as oxidative stress during herpesvirus infection has been previously documented. Unexpectedly, some oxidized proteins are removed in a proteasome-dependent fashion throughout infection and others resist degradation. Oxidized proteins that resist proteolysis become sequestered in foci within the nucleus and are not associated with virus- induced chaperone enriched domains (VICE), active centers of protein quality control, but rather coincide with Hsp27-enriched foci that were previously described by our laboratory. Experiments also indicate that the accumulation of oxidized proteins is more pronounced in cells depleted for Hsp27. We propose that Hsp27 may facilitate oxidized protein turnover at VICE domains in the nucleus during infection. Hsp27 may also buffer toxic effects of highly-carbonylated, defective proteins that resist proteolysis by promoting their aggregation in the nucleus. These roles of Hsp27 during virus infection are most likely not mutually exclusive.

Glycoprotein gene sequence variation in rhesus monkey rhadinovirus

Gene sequences for seven glycoproteins from 20 independent isolates of rhesus monkey rhadinovirus (RRV) and of the corresponding seven glycoprotein genes from nine strains of the Kaposi's sarcoma-associated herpesvirus (KSHV) were obtained and analyzed. Phylogenetic analysis revealed two discrete groupings of RRV gH sequences, two discrete groupings of RRV gL sequences and two discrete groupings of RRV gB sequences. We called these phylogenetic groupings gH(a), gH(b), gL(a), gL(b), gB(a) and gB(b). gH(a) was always paired with gL(a) and gH(b) was always paired with gL(b) for any individual RRV isolate. Since gH and gL are known to be interacting partners, these results suggest the need of matching sequence types for function of these cooperating proteins. gB phylogenetic grouping was not associated with gH/gL phylogenetic grouping. Our results demonstrate two distinct, distantly-related phylogenetic groupings of gH and gL of RRV despite a remarkable degree of sequence conservation within each individual phylogenetic group.