Functional divergence of Kaposi's sarcoma-associated herpesvirus and related gamma-2 herpesvirus thymidine kinases: novel cytoplasmic phosphoproteins that alter cellular morphology and disrupt adhesion

The nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a relatively inefficient enzyme with substrate specificity for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs). Similar to all gammaherpesvirus TKs, KSHV TK is composed of two distinct domains, a conserved C-terminal kinase and a novel and uncharacterized N terminus. Ectopic expression of KSHV TK in adherent cells induced striking morphological changes and anchorage independence although cells survived, a property shared with the related rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri. To determine whether KSHV TK served alternate functions relevant to the rhadinovirus life cycle and to reveal the contribution of the N terminus, an enhanced green fluorescent protein-tagged fusion protein and serial mutants were generated for investigation of intracellular localization and cell biology. Analysis of truncation mutants showed that a proline-rich region located within the N terminus cooperated with the conserved C-terminal kinase to tether KSHV TK to a reticular network in the cytoplasm and to induce morphological change. Fusion of the KSHV N terminus to herpes simplex virus type 1 TK, a nucleus-localized enzyme, similarly resulted in cytoplasmic redistribution of the chimeric protein but did not alter cell shape or adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK mutant that was hypophosphorylated failed to detach and grow in suspension. Loss of adhesion may enhance terminal differentiation, viral replication, and egress at the cellular level and at the organism level may facilitate detachment and distant migration of KSHV-replicating cells within body fluids--promoting oropharyngeal transmission and perhaps contributing to the multifocal lesions that characterize KS.     

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.     

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.     

Sequence and genomic analysis of a Rhesus macaque rhadinovirus with similarity to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8

We have sequenced the long unique region (LUR) and characterized the terminal repeats of the genome of a rhesus rhadinovirus (RRV), strain 17577. The LUR as sequenced is 131,364 bp in length, with a G+C content of 52.2% and a CpG ratio of 1.11. The genome codes for 79 open reading frames (ORFs), with 67 of these ORFs similar to genes found in both Kaposi's sarcoma-associated herpesvirus (KSHV) (formal name, human herpesvirus 8) and herpesvirus saimiri. Eight of the 12 unique genes show similarity to genes found in KSHV, including genes for viral interleukin-6, viral macrophage inflammatory protein, and a family of viral interferon regulatory factors (vIRFs). Genomic organization is essentially colinear with KSHV, the primary differences being the number of cytokine and IRF genes and the location of the gene for dihydrofolate reductase. Highly repetitive sequences are located in positions corresponding to repetitive sequences found in KSHV. Phylogenetic analysis of several ORFs supports the similarity between RRV and KSHV. Overall, the sequence, structural, and phylogenetic data combine to provide strong evidence that RRV 17577 is the rhesus macaque homolog of KSHV.     

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.     

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.     

Analysis of 4.3 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi's sarcoma-associated herpesvirus

We previously identified retroperitoneal fibromatosis-associated herpesvirus (RFHV) as a simian homolog of Kaposi's sarcoma-associated herpesvirus (KSHV) in a fibroproliferative malignancy of macaques that has similarities to Kaposi's sarcoma. In this report, we cloned 4.3 kb of divergent locus B (DL-B) flanking the DNA polymerase gene from two variants of RFHV from different species of macaque with a consensus degenerate hybrid oligonucleotide primer approach. Within the DL-B region of RFHV, viral homologs of the cellular interleukin-6, dihydrofolate reductase, and thymidylate synthase genes were identified, along with a homolog of the gammaherpesvirus open reading frame (ORF) 10. In addition, a homolog of the KSHV ORF K3, the modulator of immune recognition-1, was identified. Our data show a close similarity in sequence conservation, gene content, and genomic structure between RFHV and KSHV which strongly supports the grouping of these viral species within the same RV-1 rhadinovirus lineage and the hypothesis that RFHV is the macaque homolog of KSHV.     

Feeling manipulated: cytomegalovirus immune manipulation

Background

ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication.

Results

We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses.

Conclusion

The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.     

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.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

Mass spectrometric analyses of purified rhesus monkey rhadinovirus reveal 33 virion-associated proteins

The repertoire of proteins that comprise intact gammaherpesviruses, including the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is likely to have critical functions not only in viral structure and assembly but also in the early stages of infection and evasion of the host's rapidly deployed antiviral defenses. To develop a better understanding of these proteins, we analyzed the composition of rhesus monkey rhadinovirus (RRV), a close phylogenetic relative of KSHV. Unlike KSHV, RRV replicates to high titer in cell culture and thus serves as an effective model for studying primate gammaherpesvirus structure and virion proteomics. We employed two complementary mass spectrometric approaches and found that RRV contains at least 33 distinct virally encoded proteins. We have assigned 7 of these proteins to the capsid, 17 to the tegument, and 9 to the envelope. Of the five gammaherpesvirus-specific tegument proteins, three have no known function. We also found three proteins not previously associated with a purified herpesvirus and an additional seven that represent new findings for a member of the gamma-2 herpesviruses. Detergent extraction resulted in particles that contained six distinct tegument proteins in addition to the expected capsid structural proteins, suggesting that this subset of tegument components may interact more directly with or with higher affinity for the underlying capsid and, in turn, may play a role in assembly or transport of viral or subviral particles during entry or egress.     

Recent advances in the study of Kaposi’s sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi’s sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi’s sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 (HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

KSHV infection of B-cell lymphoma using a modified KSHV BAC36 and coculturing system

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV infection of B cell lymphoma in vitro has been a long-standing battle in advancing human KSHV biology. In this study, a modified form of KSHV BAC36 named BAC36A significantly increased the fidelity of gene-targeted site-directed mutagenesis in the KSHV genome. This modification eliminates tedious screening steps required to obtain mutant clones when a KSHV BAC36 reverse genetic system is used. Coculturing B-cell lymphoma BJAB cells with KSHV BAC36A stably transfected 293T cells enabled us to infect BJAB cells with a KSHV virion derived from the KSHV BAC36A. The coculture system produced substantial amounts of KSHV infection to BJAB, meaning that KSHV virions were released from 293T cells and then infected neighboring BJAB cells. Owing to our success with the KSHV BAC36A and coculture system, we propose a new genetic system for the study of KSHV gene expression and regulation in B-cell lymphoma.     

Species specificity of macaque rhadinovirus glycoprotein B sequences

All members of the Herpesviridae family contain sequences for a highly conserved glycoprotein B (gB) gene. We investigated the phylogenetic relationships of gB sequences from eight independent rhadinovirus isolates obtained from three species: rhesus (Macaca mulatta), cynomologus (Macaca fasicularis), and pig-tailed (Macaca nemestrina) macaques. Samples were derived from monkeys housed at four separate facilities. Analysis of these eight independent gB sequences revealed five regions of heterogeneity within the 823- to 829-amino-acid polypeptides: residues 1 to 65, 120 to 185, 255 to 300, 352 to 393, and 412 to 457. The remaining regions of gB were highly conserved among the different macaque isolates. Overall divergence among these gene sequences ranged from 0.1 to 7.2% at the amino acid level. Phylogenetic trees constructed with our macaque rhadinovirus gB sequences and those derived from additional subfamilies or genera (alpha, beta, gamma-1, and gamma-2) revealed that the macaque gB sequences branched with other gamma-2 herpesvirus gB sequences and that within the gamma-2 genera, the macaque gB sequences clustered as a distinct branch. The eight macaque rhadinovirus gB sequences were all approximately equidistant from Kaposi sarcoma-associated herpesvirus (KSHV) gB sequences and had a shorter evolutionary distance to KSHV gB sequences than to any other herpesvirus, including the gamma-2 herpesvirus saimiri (HVS) of New World squirrel monkeys. The macaque gB sequences did not cluster according to the facility of origin, but did cluster according to the species of origin, displaying less intraspecies divergence (0.1 to 2.9%) than interspecies divergence (3.3 to 7.2%). These results demonstrate a close relatedness of rhadinovirus isolates from different macaque species.