Quantitative RNAseq analysis of Ugandan KS tumors reveals KSHV gene expression dominated by transcription from the LTd downstream latency promoter.

KSHV is endemic in Uganda and the HIV epidemic has dramatically increased the incidence of Kaposi sarcoma (KS). To investigate the role of KSHV in the development of KS, we obtained KS biopsies from ART-naïve, HIV-positive individuals in Uganda and analyzed the tumors using RNAseq to globally characterize the KSHV transcriptome. Phylogenetic analysis of ORF75 sequences from 23 tumors revealed 6 distinct genetic clusters with KSHV strains exhibiting M, N or P alleles. RNA reads mapping to specific unique coding sequence (UCDS) features were quantitated using a gene feature file previously developed to globally analyze and quantitate KSHV transcription in infected endothelial cells. A pattern of high level expression was detected in the KSHV latency region that was common to all KS tumors. The clear majority of transcription was derived from the downstream latency transcript promoter P3(LTd) flanking ORF72, with little evidence of transcription from the P1(LTc) latency promoter, which is constitutive in KSHV-infected lymphomas and tissue-culture cells. RNAseq data provided evidence of alternate P3(LTd) transcript editing, splicing and termination resulting in multiple gene products, with 90% of the P3(LTd) transcripts spliced to release the intronic source of the microRNAs K1-9 and 11. The spliced transcripts encode a regulatory uORF upstream of Kaposin A with alterations in intervening repeat sequences yielding novel or deleted Kaposin B/C-like sequences. Hierarchical clustering and PCA analysis of KSHV transcripts revealed three clusters of tumors with different latent and lytic gene expression profiles. Paradoxically, tumors with a latent phenotype had high levels of total KSHV transcription, while tumors with a lytic phenotype had low levels of total KSHV transcription. Morphologically distinct KS tumors from the same individual showed similar KSHV gene expression profiles suggesting that the tumor microenvironment and host response play important roles in the activation level of KSHV within the infected tumor cells.

Experimental co-transmission of Simian Immunodeficiency Virus (SIV) and the macaque homologs of the Kaposi Sarcoma-Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV).

Macaque RFHV and LCV are close homologs of human KSHV and EBV, respectively. No experimental model of RFHV has been developed due to the lack of a source of culturable infectious virus. Screening of macaques at the Washington National Primate Research Center detected RFHV in saliva of SIV-infected macaques from previous vaccine studies. A pilot experimental infection of two naïve juvenile pig-tailed macaques was initiated by inoculation of saliva from SIV-infected pig-tailed and cynomolgus macaque donors, which contained high levels of DNA (> 10(6) genomes/ml) of the respective species-specific RFHV strain. Both juvenile recipients developed SIV and RFHV infections with RFHV DNA detected transiently in saliva and/or PBMC around week 16 post-infection. One juvenile macaque was infected with the homologous RFHVMn from whole saliva of a pig-tailed donor, which had been inoculated into the cheek pouch. This animal became immunosuppressed, developing simian AIDS and was euthanized 23 weeks after inoculation. The levels of RFHV DNA in saliva and PBMC remained below the level of detection after week 17, showing no reactivation of the RFHVMn infection during the rapid development of AIDS. The other juvenile macaque was infected with the heterologous RFHVMf from i.v. inoculation of purified virions from saliva of a cynomolgus donor. The juvenile recipient remained immunocompetent, developing high levels of persistent anti-RFHV and -SIV antibodies. After the initial presence of RFHVMf DNA in saliva and PBMC decreased to undetectable levels by week 19, all attempts to reactivate the infection through additional inoculations, experimental infection with purified SRV-2 or SIV, or immunosuppressive treatments with cyclosporine or dexamethasone were unsuccessful. An heterologous LCV transmission was also detected in this recipient, characterized by continual high levels of LCVMf DNA from the cynomolgus donor in both saliva (> 10(6) genomes/ml) and PBMC (> 10(4) genomes/million cells), coupled with high levels of anti-LCV antibodies. The macaque was sacrificed 209 weeks after the initial inoculation. Low levels of LCVMf DNA were detected in salivary glands, tonsils and other lymphoid organs, while RFHVMf DNA was below the level of detection. These results show successful co-transmission of RFHV and LCV from saliva and demonstrate differential lytic activation of the different gammaherpesvirus lineages due to presumed differences in biology and tropism and control by the host immune system. Although this initial pilot transmission study utilized only two macaques, it provides the first evidence for experimental transmission of the macaque homolog of KSHV, setting the stage for larger transmission studies to examine the differential activation of rhadinovirus and lymphocryptovirus infections and the pathological effects of immunosuppression.

Base-By-Base Version 3: New Comparative Tools for Large Virus Genomes.

Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of "consensus-degenerate hybrid oligonucleotide primers" (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.

Macaque homologs of Kaposi's sarcoma-associated herpesvirus (KSHV) infect germinal center lymphoid cells, epithelial cells in skin and gastrointestinal tract and gonadal germ cells in naturally infected macaques.

We developed a set of rabbit antisera to characterize infections by the macaque RV2 rhadinovirus homologs of KSHV. We analyzed tissues from rhesus and pig-tailed macaques naturally infected with rhesus rhadinovirus (RRV) or Macaca nemestrina rhadinovirus 2 (MneRV2). Our study demonstrates that RV2 rhadinoviruses have a tropism for epithelial cells, lymphocytes and gonadal germ cells in vivo. We observed latent infections in both undifferentiated and differentiated epithelial cells with expression of the latency marker, LANA. Expression of the early (ORF59) and late (glycoprotein B) lytic markers were detected in highly differentiated cells in epithelial ducts in oral, renal, dermal and gastric mucosal tissue as well as differentiated germ cells in male and female gonads. Our data provides evidence that epithelial and germ cell differentiation in vivo induces rhadinovirus reactivation and suggests that infected epithelial and germ cells play a role in transmission and dissemination of RV2 rhadinovirus infections in vivo.

KSHV oral shedding and plasma viremia result in significant changes in the extracellular tumorigenic miRNA expression profile in individuals infected with the malaria parasite.

Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS). Both KSHV and HIV infections are endemic in Uganda, where KS is among the most common cancers in HIV-infected individuals. Recent studies examined the use of small RNAs as biomarkers of disease, including microRNAs (miRNAs), with viral and tumor-derived miRNAs being detected in exosomes from individuals with KSHV-associated malignancies. In the current study, the host and viral extracellular mature miRNA expression profiles were analyzed in blood of KS-negative individuals in Uganda, comparing those with or without KSHV detectable from the oropharynx. We observed increased levels of cellular oncogenic miRNAs and decreased levels of tumor-suppressor miRNAs in plasma of infected individuals exhibiting oral KSHV shedding. These changes in host oncomiRs were exacerbated in people co-infected with HIV, and partially reversed after 2 years of anti-retroviral therapy. We also detected KSHV miRNAs in plasma of KSHV infected individuals and determined that their expression levels correlated with KSHV plasma viremia. Deep sequencing revealed an expected profile of small cellular RNAs in plasma, with miRNAs constituting the major RNA biotype. In contrast, the composition of small RNAs in exosomes was highly atypical with high levels of YRNA and low levels of miRNAs. Mass spectrometry analysis of the exosomes revealed eleven different peptides derived from the malaria parasite, Plasmodium falciparum, and small RNA sequencing confirmed widespread plasmodium co-infections in the Ugandan cohorts. Proteome analysis indicated an exosomal protein profile consistent with erythrocyte and keratinocyte origins for the plasma exosomes. A strong correlation was observed between the abundance of Plasmodium proteins and cellular markers of malaria. As Plasmodium falciparum is an endemic pathogen in Uganda, our study shows that co-infection with other pathogens, such as KSHV, can severely impact the small RNA repertoire, complicating the use of exosome miRNAs as biomarkers of disease.

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication.

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin ß1 for non-classical nuclear import.

The latency-associated nuclear antigens (LANA) of KSHV and macaque RFHVMn, members of the RV1 rhadinovirus lineage, are closely related with conservation of complex nuclear localization signals (NLS) containing bipartite KR-rich motifs and RG-rich domains, which interact distinctly with importins α and ß1 for nuclear import via classical and non-classical pathways, respectively. RV1 LANAs are expressed in the nucleus of latently-infected cells where they inhibit replication and establish a dominant RV1 latency. Here we show that LANA homologs of macaque RRV and MneRV2 from the more distantly-related RV2 lineage, lack the KR-rich NLS, and instead have a large RG-rich NLS with multiple RG dipeptides and a conserved RGG motif. The RG-NLS interacts uniquely with importin ß1, which mediates nuclear import and accumulation of RV2 LANA in the nucleolus. The alternative nuclear import and localization of RV2 LANA homologs may contribute to the dominant RV2 lytic replication phenotype.

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells.

The transcriptome of the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial induction, and a comprehensive annotation of the KSHV genome was determined. A set of unique coding sequence (UCDS) features and a process to resolve overlapping transcripts were developed to accurately quantitate transcript levels from specific promoters. Similar patterns of KSHV expression were detected in BCBL-1 cells undergoing long-term latent infections and in primary latent infections of both BEC and LEC cultures. High expression levels of poly-adenylated nuclear (PAN) RNA and spliced and unspliced transcripts encoding the K12 Kaposin B/C complex and associated microRNA region were detected, with an elevated expression of a large set of lytic genes in all latently infected cultures. Quantitation of non-overlapping regions of transcripts across the complete KSHV genome enabled for the first time accurate evaluation of the KSHV transcriptome associated with viral latency in different cell types. Hierarchical clustering applied to a gene correlation matrix identified modules of co-regulated genes with similar correlation profiles, which corresponded with biological and functional similarities of the encoded gene products. Gene modules were differentially upregulated during latency in specific cell types indicating a role for cellular factors associated with differentiated and/or proliferative states of the host cell to influence viral gene expression.

Conservation of the glycoprotein B homologs of the Kaposi׳s sarcoma-associated herpesvirus (KSHV/HHV8) and old world primate rhadinoviruses of chimpanzees and macaques.

The envelope-associated glycoprotein B (gB) is highly conserved within the Herpesviridae and plays a critical role in viral entry. We analyzed the evolutionary conservation of sequence and structural motifs within the Kaposi׳s sarcoma-associated herpesvirus (KSHV) gB and homologs of Old World primate rhadinoviruses belonging to the distinct RV1 and RV2 rhadinovirus lineages. In addition to gB homologs of rhadinoviruses infecting the pig-tailed and rhesus macaques, we cloned and sequenced gB homologs of RV1 and RV2 rhadinoviruses infecting chimpanzees. A structural model of the KSHV gB was determined, and functional motifs and sequence variants were mapped to the model structure. Conserved domains and motifs were identified, including an "RGD" motif that plays a critical role in KSHV binding and entry through the cellular integrin αVß3. The RGD motif was only detected in RV1 rhadinoviruses suggesting an important difference in cell tropism between the two rhadinovirus lineages.

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.

UNLABELLED: 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. IMPORTANCE: 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.

KSHV attachment and entry are dependent on αVß3 integrin localized to specific cell surface microdomains and do not correlate with the presence of heparan sulfate.

Cellular receptors for KSHV attachment and entry were characterized using tyramide signal amplification (TSA)-enhanced confocal microscopy. Integrins αVß3, αVß5 and α3ß1 were detected on essentially all the actin-based cell surface microdomains that initially bind KSHV, while the presence of CD98 and heparan sulfate (HS), the putative attachment receptor, was more variable. KSHV bound to the same cell surface microdomains with and without HS indicating that initial attachment of KSHV is not dependent on HS and that receptors other than HS can mediate attachment. A human salivary gland (HSG) epithelial line was identified, which lacks αVß3 but expresses high levels of HS, α3ß1 and other putative KSHV receptors. These cells were resistant to KSHV binding and infection. Reconstitution of cell surface αVß3 rendered HSG cells highly susceptible to KSHV infection, demonstrating a critical role for αVß3 in the binding and entry of KSHV that is not shared with other proposed receptors.

Oral squamous cell carcinoma in a pigtailed macaque (Macaca nemestrina).

An adult, gravid, female pigtailed macaque (Macaca nemestrina) presented for facial swelling centered on the left mandible that was approximately 5 cm wide. Differential diagnoses included infectious, inflammatory, and neoplastic origins. Definitive antemortem diagnosis was not possible, and the macaque's condition worsened despite supportive care. Necropsy findings included a mandibular mass that was locally invasive and expansile, encompassing approximately 80% of the left mandibular bone. The mass replaced portions of the soft palate, hard palate, sinuses, ear canal, and the caudal-rostral calvarium and masseter muscle. Histologically, the mass was a neoplasm that was poorly circumscribed, unencapsulated, and infiltrative invading regional bone and soft tissue. The mass consisted of polygonal squamous epithelial cells with intercellular bridging that breached the epithelial basement membrane and formed invasive nests, cords, and trabeculae. The mitotic rate averaged 3 per 400× field of view, with occasional bizarre mitotic figures. Epithelial cells often exhibited dyskeratosis, and the nests often contained compact lamellated keratin (keratin pearls). The neoplasm was positive via immunohistochemistry for pancytokeratin, variably positive for S100, and negative for vimentin, smooth muscle actin, and desmin. The gross, histologic, and immunohistochemical findings were consistent with an aggressive oral squamous cell carcinoma. The neoplasm was negative via PCR for papilloma virus. In general, neoplasia in macaques is rare. Although squamous cell carcinomas are one of the most common oral neoplasia in many species, to our knowledge this case represents the first reported oral squamous cell carcinoma in a pigtailed macaque.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry.

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.

Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

Next-generation sequence analysis of the genome of RFHVMn, the macaque homolog of Kaposi's sarcoma (KS)-associated herpesvirus, from a KS-like tumor of a pig-tailed macaque.

The complete sequence of retroperitoneal fibromatosis-associated herpesvirus Macaca nemestrina (RFHVMn), the pig-tailed macaque homolog of Kaposi's sarcoma-associated herpesvirus (KSHV), was determined by next-generation sequence analysis of a Kaposi's sarcoma (KS)-like macaque tumor. Colinearity of genes was observed with the KSHV genome, and the core herpesvirus genes had strong sequence homology to the corresponding KSHV genes. RFHVMn lacked homologs of open reading frame 11 (ORF11) and KSHV ORFs K5 and K6, which appear to have been generated by duplication of ORFs K3 and K4 after the divergence of KSHV and RFHV. RFHVMn contained positional homologs of all other unique KSHV genes, although some showed limited sequence similarity. RFHVMn contained a number of candidate microRNA genes. Although there was little sequence similarity with KSHV microRNAs, one candidate contained the same seed sequence as the positional homolog, kshv-miR-K12-10a, suggesting functional overlap. RNA transcript splicing was highly conserved between RFHVMn and KSHV, and strong sequence conservation was noted in specific promoters and putative origins of replication, predicting important functional similarities. Sequence comparisons indicated that RFHVMn and KSHV developed in long-term synchrony with the evolution of their hosts, and both viruses phylogenetically group within the RV1 lineage of Old World primate rhadinoviruses. RFHVMn is the closest homolog of KSHV to be completely sequenced and the first sequenced RV1 rhadinovirus homolog of KSHV from a nonhuman Old World primate. The strong genetic and sequence similarity between RFHVMn and KSHV, coupled with similarities in biology and pathology, demonstrate that RFHVMn infection in macaques offers an important and relevant model for the study of KSHV in humans.

Development of whole-virus multiplex luminex-based serological assays for diagnosis of infections with kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 homologs in macaques.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi's sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.