Three-Dimensional Structures of the A, B, and C Capsids of Rhesus Monkey Rhadinovirus: Insights into Gammaherpesvirus Capsid Assembly, Maturation, and DNA Packaging

Rhesus monkey rhadinovirus (RRV) exhibits high levels of sequence homology to human gammaherpesviruses, such as Kaposi's sarcoma-associated herpesvirus, and grows to high titers in cell cultures, making it a good model system for studying gammaherpesvirus capsid structure and assembly. We have purified RRV A, B, and C capsids, thus for the first time allowing direct structure comparisons by electron cryomicroscopy and three-dimensional reconstruction. The results show that the shells of these capsids are identical and are each composed of 12 pentons, 150 hexons, and 320 triplexes. Structural differences were apparent inside the shells and through the penton channels. The A capsid is empty, and its penton channels are open. The B capsid contains a scaffolding core, and its penton channels are closed. The C capsid contains a DNA genome, which is closely packaged into regularly spaced density shells (25 Å apart), and its penton channels are open. The different statuses of the penton channels suggest a functional role of the channels during capsid maturation, and the overall structural similarities of RRV capsids to alphaherpesvirus capsids suggest a common assembly and maturation pathway. The RRV A capsid reconstruction at a 15-Å resolution, the best achieved for gammaherpesvirus particles, reveals overall structural similarities to alpha- and betaherpesvirus capsids. However, the outer regions of the capsid, including densities attributed to the Ta triplex and the small capsomer-interacting protein (SCIP or ORF65), exhibit prominent differences from their structural counterparts in alphaherpesviruses. This structural disparity suggests that SCIP and the triplex, together with tegument and envelope proteins, confer structural and potentially functional specificities to alpha-, beta-, and gammaherpesviruses

Risk factors for high anti-HHV-8 antibody titers (≥1:51,200) in black, HIV-1 negative South African cancer patients: a case control study

Background: Infection with human herpesvirus 8 (HHV-8) is the necessary causal agent in the development of Kaposi's sarcoma (KS). Infection with HIV-1, male gender and older age all increase risk for KS. However, the geographic distribution of HHV-8 and KS both prior to the HIV/AIDS epidemic and with HIV/AIDS suggest the presence of an additional co-factor in the development of KS. Methods: Between January 1994 and October 1997, we interviewed 2576 black in-patients with cancer in Johannesburg and Soweto, South Africa. Blood was tested for antibodies against HIV-1 and HHV-8 and the study was restricted to 2191 HIV-1 negative patients. Antibodies against the latent nuclear antigen of HHV-8 encoded by orf73 were detected with an indirect immunofluorescence assay. We examined the relationship between high anti-HHV-8 antibody titers (≥1:51,200) and sociodemographic and behavioral factors using unconditional logistic regression models. Variables that were significant at p = 0.10 were included in multivariate analysis. Results: Of the 2191 HIV-1 negative patients who did not have Kaposi's sarcoma, 854 (39.0%) were positive for antibodies against HHV-8 according to the immunofluorescent assay. Among those seropositive for HHV-8, 530 (62.1%) had low titers (1:200), 227 (26.6%) had medium titers (1:51,200) and 97 (11.4%) had highest titers (1:204,800). Among the 2191 HIV-1 negative patients, the prevalence of high anti-HHV-8 antibody titers (≥1:51,200) was independently associated with increasing age (ptrend = 0.04), having a marital status of separated or divorced (p = 0.003), using wood, coal or charcoal as fuel for cooking 20 years ago instead of electricity (p = 0.02) and consuming traditional maize beer more than one time a week (p = 0.02; p-trend for increasing consumption = 0.05) although this may be due to chance given the large number of predictors considered in this analysis. Conclusions: Among HIV-negative subjects, patients with high anti-HHV-8 antibody titers are characterized by older age. Other associations that may be factors in the development of high anti- HHV-8 titers include exposure to poverty or a low socioeconomic status environment and consumption of traditional maize beer. The relationship between these variables and high anti- HHV-8 titers requires further, prospective study

Seroprevalence of human herpesvirus-8 (HHV-8) in countries of Southeast Asia compared to the USA, the Caribbean and Africa

Seroprevalence of HHV-8 has been studied in Malaysia, India, Sri Lanka, Thailand, Trinidad, Jamaica and the USA, in both healthy individuals and those infected with HIV. Seroprevalence was found to be low in these countries in both the healthy and the HIV-infected populations. This correlates with the fact that hardly any AIDS-related Kaposi’s sarcoma has been reported in these countries. In contrast, the African countries of Ghana, Uganda and Zambia showed high seroprevalences in both healthy and HIV-infected populations. This suggests that human herpes virus-8 (HHV-8) may be either a recently introduced virus or one that has extremely low infectivity. Nasopharyngeal and oral carcinoma patients from Malaysia, Hong Kong and Sri Lanka who have very high EBV titres show that only 3/82 (3.7%) have antibody to HHV-8, demonstrating that there is little, if any, cross-reactivity between antibodies to these two gamma viruses. © 1999 Cancer Research Campaig

Seroprevalence and risk factors of Kaposi's sarcoma-associated herpesvirus infection among the general Uygur population from south and north region of Xinjiang, China

<p>Abstract</p> <p>Background</p> <p>Kaposi sarcoma (KS) is a complex multifocal neoplasm and is the major cause of death for about 50% of acquired immunodeficiency syndrome (AIDS) patients. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus with a causal role in the development of all types of KS. KS is prevalent among the Uygur people in Xinjiang, especially in south area. Here we carried out a cross-sectional study among 1534 general Uygur individuals from south and north region of Xinjiang to assess the seroprevalence of KSHV and to identify the potential correlation between KSHV seroprevalence and KS incidence.</p> <p>Results</p> <p>Seroprevalence of KSHV in South and North Xinjiang was 23.1% and 25.9%, respectively. Older age was independently associated with higher KSHV seroprevalence. In subjects from South Xinjiang, lower educational level and reported drinking were each independently associated with higher KSHV seroprevalence. Furthermore, the antibody titer was significantly lower in both south and north KSHV seropositive individuals compared with KS patients, as analyzed by gradient dilution (P < 0.001).</p> <p>Conclusion</p> <p>KSHV is highly prevalent in the general Uygur population in both South and North Xinjiang. Interestingly, the infection rate of KSHV in these two geographical areas did not correlate well with KS incidence. Perhaps unknown factors exist that promote the progression of KSHV infection to KS development in the local minority groups.</p

Rapamycin Blocks Production of KSHV/HHV8: Insights into the Anti-Tumor Activity of an Immunosuppressant Drug

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) often results in the development of fatal tumors in immunocompromised patients. Studies of renal transplant recipients show that use of the immunosuppressant drug rapamycin, an mTOR inhibitor, both prevents and can induce the regression of Kaposi's sarcoma (KS), an opportunistic tumor that arises within a subset of this infected population. In light of rapamycin's marked anti-KS activity, we tested whether the drug might directly inhibit the KSHV life cycle. We focused on the molecular switch that triggers this predominantly latent virus to enter the lytic (productive) replication phase, since earlier work links this transition to viral persistence and tumorigenesis.In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA). To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition. Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation. Finally, treatment with rapamycin blocked the production of progeny virions.These results indicate that mTOR plays a role in the regulation of RTA expression and, therefore, KSHV production, providing a potential molecular explanation for the marked clinical success of rapamycin in the treatment and prevention of post-transplant Kaposi's sarcoma. The striking inhibition of rapamycin on KSHV lytic replication, thus, helps explain the apparent paradox of an immunosuppressant drug suppressing the pathogenesis of an opportunistic viral infection

Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae

<p>Abstract</p> <p>Background</p> <p>All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga <it>Cyanidioschyzon merolae </it>revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the <it>C. merolae </it>genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.</p> <p>Results</p> <p>Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the <it>C. merolae </it>genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.</p> <p>Conclusion</p> <p>By virtue of these attributes and others that we had discovered previously, <it>C. merolae </it>appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of <it>C. merolae </it>are extremely useful for further studies of eukaryotic cells.</p

KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA

Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA–mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus–host interactions during reactivation and may represent potential novel targets for therapeutic intervention

Conservation of Complex Nuclear Localization Signals Utilizing Classical and Non-Classical Nuclear Import Pathways in LANA Homologs of KSHV and RFHV

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency

Opposing Regulation of PROX1 by Interleukin-3 Receptor and NOTCH Directs Differential Host Cell Fate Reprogramming by Kaposi Sarcoma Herpes Virus

Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV