Factors affecting Kaposi's sarcoma-associated herpesvirus transmission in rural Ugandan households, a longitudinal study.

BACKGROUND: We report the impact of HIV infection within a household on oral Kaposi's sarcoma-associated herpesvirus (KSHV) shedding. METHODS: We enrolled 469 individuals from 90 households. Mouthwash rinse samples collected at three monthly visits were analyzed for KSHV DNA using quantitative polymerase chain reaction (qPCR). Generalized linear mixed effects logistic models were applied to analyze factors associated with KSHV ever shedding, and among shedders, always versus intermittent shedding. Linear mixed effects models were applied to models of KSHV viral loads. Intraclass correlation coefficients (ICCs) were calculated to assess the contribution of household-level factors to variations in shedding probabilities. Hotspot analyses of geospatial feature clusters were calculated using Getis-Ord Gi* statistic and visualized using inverse distance weighted interpolation. RESULTS: Analyses included 340 KSHV seropositive individuals, aged 3 + years, with qPCR results from 89 households. Forty households had 1 + persons living with HIV (PLWH), while 49 had none. Among participants, 149(44%) were KSHV ever shedders. Of 140 who shed KSHV at two or more visits, 34(24%) were always shedders. Increasing number of KSHV seropositive household members was significantly associated with ever shedding [Odds ratio(OR) (95% Confidence Interval(95%CI)):1.14(1.03,1.26);p = 0.013]. Among KSHV shedders, a statistically significant age-related trend was identified with 10-19 years being more likely to be always shedders (type III test p = 0.039) and to have higher viral loads (type III test p = 0.027). In addition, higher viral loads were significantly associated with increasing number of household members [coefficient(95%CI):0.06(0.01,0.12);p = 0.042], increasing number of KSHV seropositive members [coefficient(95%CI):0.08(0.01,0.15);p = 0.021], and living in households with 1 + PLWH [coefficient(95%CI):0.51(0.04,0.98);p = 0.033]. Always shedders exhibited higher viral loads than intermittent shedders [coefficient(95%CI):1.62(1.19,2.05);p < 0.001], and viral loads increased with the number of visits where KSHV DNA was detected in saliva (type III test p < 0.001). Household-level factors attributed for 19% of the variability in KSHV shedding (ICC:0.191;p = 0.010). Geospatial analysis indicated overlapping hotspots of households with more KSHV seropositive individuals and KSHV shedders, distinct from areas where PLWH were clustered. DISCUSSION: KSHV oral shedding is influenced by multiple factors at the individual, household, and regional levels. To mitigate ongoing KSHV transmission a comprehensive understanding of factors contributing to oral KSHV reactivation and transmission within households is needed.

Highly Heterogeneous Kaposi Sarcoma-Associated Herpesvirus Oral Shedding Kinetics Among People With and Without Kaposi Sarcoma and Human Immunodeficiency Virus Coinfection.

Background: An improved understanding of oral Kaposi sarcoma-associated herpesvirus (KSHV) viral dynamics could provide insights into transmission risk and guide vaccine development. Methods: We evaluated KSHV oral shedding dynamics in Ugandan adults stratified by Kaposi sarcoma (KS) and human immunodeficiency virus (HIV) status. Participants were followed for ≥4 weeks, with daily home oral swab collection to quantify KSHV using polymerase chain reaction. Shedding rates were defined by number of days with KSHV DNA detected divided by total days with swabs and compared by group using hurdle models. Results: Two hundred ninety-five participants were enrolled; median age was 35 years (range, 18-71 years), and 134 (45%) were male. KSHV was detected more frequently among participants with KS (HIV positive [HIV+]/KS+, 56/76 [74%]; HIV negative [HIV-]/KS+, 9/18 [50%]) than those without KS (HIV+/KS-, 36/125 [29%]; HIV-/KS-, 16/76 [21%]); odds of shedding did not differ significantly by HIV status. Among participants with KSHV detected, shedding rates did not differ significantly by group. Median per-participant viral loads among positive samples were lowest in HIV+/KS+ (3.1 log10 copies/mL) and HIV-/KS+ (3.3 log10 copies/mL) participants relative to HIV+/KS- (3.8 log10 copies/mL) and HIV-/KS- (4.0 log10 copies/mL) participants. All groups had participants with low viral load intermittent shedding and participants with high viral load persistent shedding. Within each group, individual KSHV shedding rate positively correlated with median KSHV log10 copies/mL, and episode duration positively correlated with peak viral load. Conclusions: Oral KSHV shedding is highly heterogeneous across Ugandan adults with and without KS and HIV. Persistent shedding is associated with higher median viral loads regardless of HIV and KS status.

Inhibiting KSHV replication by targeting the essential activities of KSHV processivity protein, PF-8.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are no cures for KSHV infection. KSHV establishes biphasic lifelong infections. During the lytic phase, new genomes are replicated by seven viral DNA replication proteins. The processivity factor's (PF-8) functions to tether DNA polymerase to DNA, so new viral genomes are efficiently synthesized. PF-8 self-associates, interacts with KSHV DNA replication proteins and the viral DNA. Inhibition of viral DNA replication would diminish the infection within a host and reduce transmission to new individuals. In this review we summarize PF-8 molecular and structural studies, detail the essential protein-protein and nucleic acid interactions needed for efficient lytic DNA replication, identify future areas for investigation and propose PF-8 as a promising antiviral target. Additionally, we discuss similarities that the processivity factor from Epstein-Barr virus shares with PF-8, which could promote a pan-herpesvirus antiviral therapeutic targeting strategy.

Structural insights into KSHV-GPCR constitutive activation and CXCL1 chemokine recognition.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral G protein-coupled receptor, KSHV-GPCR, that contributes to KSHV immune evasion and pathogenesis of Kaposi's sarcoma. KSHV-GPCR shares a high similarity with CXC chemokine receptors CXCR2 and can be activated by selected chemokine ligands. Like other herpesvirus-encoded GPCRs, KSHV-GPCR is characterized by its constitutive activity by coupling to various G proteins. We investigated the structural basis of ligand-dependent and constitutive activation of KSHV-GPCR, obtaining high-resolution cryo-EM structures of KSHV-GPCR-Gi complexes with and without the bound CXCL1 chemokine. Analysis of the apo-KSHV-GPCR-Gi structure (2.81 Å) unraveled the involvement of extracellular loop 2 in constitutive activation of the receptor. In comparison, the CXCL1-bound KSHV-GPCR-Gi structure (3.01 Å) showed a two-site binding mode and provided detailed information of CXCL1 binding to a chemokine receptor. The dual activation mechanism employed by KSHV-GPCR represents an evolutionary adaptation for immune evasion and contributes to the pathogenesis of Kaposi's sarcoma. Together with results from functional assays that confirmed the structural models, these findings may help to develop therapeutic strategies for KSHV infection.

Comparison of the indirect immunofluorescence assay and a commercial ELISA to detect KSHV antibodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus involved in several diseases. The gold standard for KSHV sero diagnosis remains the indirect immunofluorescence assay (IFA), which is time-consuming and operator-dependent. We compared this method with an enzyme-linked immunosorbent assay (ELISA) targeting solubilized KSHV whole-genome extract among positive (n = 49, including 76% of HIV-infected patients) and negative (n = 14) control groups. We also included 14 sera with equivocal IFA results. ELISA showed better performance in detecting KSHV antibodies (McNemar's test, P = 0.0455). The sensitivity and specificity of both methods were 79% (64-89) and 100% (66-100) for the IFA, respectively, and 94% (83-99) and 100% (66-100) for ELISA, respectively. All IFA equivocal results were either negative or positive with ELISA. ELISA is more reliable and could be a good alternative for determining KSHV serological status, particularly in the context of immunocompromised patients and equivocal serology with the IFA.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) sero status remains challenging because no perfect reference is available for the detection of KSHV antibodies. The current gold-standard method, the indirect immunofluorescence assay (IFA), has a very good specificity of close to 100%, but a lower sensitivity of around 80-85%, which decreases to 64-67% in immunocompromised patients. Additionally, this method is time-consuming and operator-dependent compared with new serological assays such as the enzyme-linked immunosorbent assay (ELISA). Thus, further research is still needed to improve KSHV sero diagnosis. Here, we compare the KSHV IgG ELISA kit assay (Advanced Biotechnologies Inc) with the gold-standard IFA, targeting the LANA-1 protein from latent BC-3 cell lines.

Are There More Human Cancer Viruses Left to Be Found?

Of the thousands of viruses infecting humans, only seven cause cancer in the general population. Tumor sequencing is now a common cancer medicine procedure, and so it seems likely that more human cancer viruses already would have been found if they exist. Here, we review cancer characteristics that can inform a dedicated search for new cancer viruses, focusing on Kaposi sarcoma herpesvirus and Merkel cell polyomavirus as the most recent examples of successful genomic and transcriptomic searches. We emphasize the importance of epidemiology in determining which cancers to examine and describe approaches to virus discovery. Barriers to virus discovery, such as novel genomes and viral suppression of messenger RNA expression, may exist that prevent virus discovery using existing approaches. Optimally virus hunting should be performed in such a way that if no virus is found, the tumor can be reasonably excluded from having an infectious etiology and new information about the biology of the tumor can be found.

Factors affecting Kaposi's sarcoma-associated herpesvirus transmission in rural Ugandan households, a longitudinal study.

Background: We report the impact of HIV infection within a household on oral Kaposi's sarcoma-associated herpesvirus (KSHV) shedding. Methods: We enrolled 469 individuals from 90 households. Mouthwash rinse samples collected at three monthly visits, were analyzed for KSHV DNA using quantitative polymerase chain reaction (qPCR). Generalized linear mixed effects logistic models were applied to analyze factors associated with KSHV ever shedding, and among shedders, always versus intermittent shedding. Linear mixed effects models were applied to models of KSHV viral loads. Intraclass correlation coefficients (ICCs) were calculated to assess the contribution of household-level factors to variations in shedding probabilities. Hotspot analyses of geospatial feature clusters were calculated using Getis-Ord Gi* statistic and visualized using inverse distance weighted interpolation. Results: Analyses included 340 KSHV seropositive individuals, aged 3 + years, with qPCR results from 89 households. Forty households had 1 + persons living with HIV (PLWH), while 49 had none. Among participants, 149(44%) were KSHV ever shedders. Of 140 who shed KSHV at two or more visits, 34(24%) were always shedders. Increasing number of KSHV seropositive household members was significantly associated with ever shedding [Odds ratio(OR) (95% Confidence Interval(95%CI)):1.14(1.03,1.26);p = 0.013]. Among KSHV shedders, a statistically significant age-related trend was identified with 10-19 years being more likely to be always shedders (type III test p = 0.039) and to have higher viral loads (type III test p = 0.027). In addition, higher viral loads were significantly associated with increasing number of household members [coefficient(95%CI):0.06(0.01,0.12);p = 0.042], increasing number of KSHV seropositive members [coefficient(95%CI):0.08(0.01,0.15);p = 0.021], and living in households with 1 + PLWH [coefficient(95%CI):0.51(0.04,0.98);p = 0.033]. Always shedders exhibited higher viral loads than intermittent shedders [coefficient(95%CI):1.62(1.19,2.05);p < 0.001], and viral loads increased with the number of visits where KSHV DNA was detected in saliva (type III test p < 0.001). Household-level factors attributed for 19% of the variability in KSHV shedding (ICC:0.191;p = 0.010). Geospatial analysis indicated overlapping hotspots of households with more KSHV seropositive individuals and KSHV shedders, distinct from areas where PLWH were clustered. Discussion: KSHV oral shedding is influenced by multiple factors at the individual, household, and regional levels. To mitigate ongoing KSHV transmission a comprehensive understanding of factors contributing to oral KSHV reactivation and transmission within households is needed.

EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells.

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

KSHV ORF20 Promotes Coordinated Lytic Reactivation for Increased Infectious Particle Production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi's sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

Discovery of a small-molecule inhibitor of KSHV lytic replication from the MMV pandemic response box.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS). KSHV is one of the oncoviruses that contribute to 1.5 million new infection-related cancer cases annually. Currently, there are no targeted therapies for KSHV-associated diseases. Through the development of a medium-throughput phenotype-based ELISA screening platform based on KSHV ORF57 protein detection, we screened the Medicines for Malaria Venture (MMV) Pandemic Response Box for non-cytotoxic inhibitors of KSHV lytic replication. MMV1645152 was identified as a promising inhibitor of KSHV lytic replication, suppressing KSHV immediate-early and late lytic gene expression and blocking the production of infectious KSHV virion particles at non-cytotoxic concentrations in cell line models of KSHV infection with or without EBV coinfection. MMV1645152 is a promising hit compound for the development of future therapeutic agents against KSHV-associated malignancies.

The cellular paraspeckle component SFPQ associates with the viral processivity factor ORF59 during lytic replication of Kaposi's Sarcoma-associated herpesvirus (KSHV).

Kaposi's sarcoma-associated herpesvirus (KSHV) relies on many cellular proteins to complete replication and generate new virions. Paraspeckle nuclear bodies consisting of core ribonucleoproteins splicing factor proline/glutamine-rich (SFPQ), Non-POU domain-containing octamer-binding protein (NONO), and paraspeckle protein component 1 (PSPC1) along with the long non-coding RNA NEAT1, form a complex that has been speculated to play an important role in viral replication. Paraspeckle bodies are multifunctional and involved in various processes including gene expression, mRNA splicing, and anti-viral defenses. To better understand the role of SFPQ during KSHV replication, we performed SFPQ immunoprecipitation followed by mass spectrometry from KSHV-infected cells. Proteomic analysis showed that during lytic reactivation, SFPQ associates with viral proteins, including ORF10, ORF59, and ORF61. These results are consistent with a previously reported ORF59 proteomics assay identifying SFPQ. To test if the association between ORF59 and SFPQ is important for replication, we first identified the region of ORF59 that associates with SFPQ using a series of 50 amino acid deletion mutants of ORF59 in the KSHV BACmid system. By performing co-immunoprecipitations, we identified the region spanning amino acids 101-150 of ORF59 as the association domain with SFPQ. Using this information, we generated a dominant negative polypeptide of ORF59 encompassing amino acids 101-150, that disrupted the association between SFPQ and full-length ORF59, and decreased virus production. Interestingly, when we tested other human herpesvirus processivity factors (EBV BMRF1, HSV-1 UL42, and HCMV UL44) by transfection of each expression plasmid followed by co-immunoprecipitation, we found a conserved association with SFPQ. These are limited studies that remain to be done in the context of infection but suggest a potential association of SFPQ with processivity factors across multiple herpesviruses.

PAX5 functions as a tumor suppressor by RB-E2F-mediated cell cycle arrest in Kaposi sarcoma-associated herpesvirus-infected primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a malignant B-cell lymphoma attributable to Kaposi sarcoma-associated herpesvirus (KSHV) infection. PEL is characterized by invasive behavior, showing recurrent effusions in body cavities. The clinical outcome and typical prognosis in patients with PEL are poor and potentially lethal. Clarification of the pathogenesis in PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. The expression of PAX5 is detected from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via its promoter methylation. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To elucidate the in vivo role of PAX5, we examined the restoration of PAX5 in a PEL mouse model. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has played a crucial role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic strategy for patients with PEL.

LKB1 suppresses KSHV reactivation and promotes primary effusion lymphoma progression.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Combined Flow-Fluorescence in situ hybridization to HHV-8 and EBV reveals the viral heterogeneity of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare B-cell non-Hodgkin lymphoma associated with Kaposi Sarcoma-associated herpesvirus (KSHV/HHV8) infection. Lymphoma cells are coinfected with Epstein-Barr virus (EBV) in 60-80% of cases. Tools allowing a reliable PEL diagnosis are lacking. This study reports PEL diagnosis in 4 patients using a Flow-Fluorescence in situ hybridization (FlowFISH) technique that allowed detection of differentially expressed EBV and HHV8 transcripts within the same sample, revealing viral heterogeneity of the disease. Moreover, infected cells exhibited variable expressions of CD19, CD38, CD40, and CD138. Therefore, FlowFISH is a promising tool to diagnose and characterize complex viral lymphoproliferations.

Molecular detection and genotyping of human herpes virus 8 in blood donors in Congo.

OBJECTIVES: Human herpesvirus 8 (HHV8) is rarely studied in Congo, despite its prevalence in Africa. Among healthy individuals, HHV-8 does not always lead to a life-threatening infection; however, in immunocompromised individuals, it could lead to more severe disease. The distribution of HHV-8 genotypes varies depending on ethnicity and geographic region. METHOD: A prospective cross-sectional study included 265 samples from healthy blood donors from the National Blood Transfusion Center in Brazzaville, with an average age of 35 years, with extremes ranging from 18 to 60 years. After DNA extraction, a nested PCR was carried out for molecular detection, followed by genotyping by amplification of specific primers. RESULT: In this study, 4.9% were positive for molecular detection of HHV-8 DNA. All HHV-8 positive DNA samples that were subjected to genotyping by amplification with specific primers allowing discrimination of two major genotypes (A and B). Genotype A was identified in 5 (1.9%) samples and genotype B in 2 (0.7%) samples, indicating that both genotypes were predominant. The remaining viral DNA samples not identified as the major genotypes were classified as «indeterminate¼ and consisted of 6 (2.3%) samples. CONCLUSION: The results of the study suggest that Congo is an area where HHV-8 infection is endemic.

HIV-associated cancers and lymphoproliferative disorders caused by Kaposi sarcoma herpesvirus and Epstein-Barr virus.

SUMMARYWithin weeks of the first report of acquired immunodeficiency syndrome (AIDS) in 1981, it was observed that these patients often had Kaposi sarcoma (KS), a hitherto rarely seen skin tumor in the USA. It soon became apparent that AIDS was also associated with an increased incidence of high-grade lymphomas caused by Epstein-Barr virus (EBV). The association of AIDS with KS remained a mystery for more than a decade until Kaposi sarcoma-associated herpesvirus (KSHV) was discovered and found to be the cause of KS. KSHV was subsequently found to cause several other diseases associated with AIDS and human immunodeficiency virus (HIV) infection. People living with HIV/AIDS continue to have an increased incidence of certain cancers, and many of these cancers are caused by EBV and/or KSHV. In this review, we discuss the epidemiology, virology, pathogenesis, clinical manifestations, and treatment of cancers caused by EBV and KSHV in persons living with HIV.

Nontypeable Haemophilus influenzae challenge during gammaherpesvirus infection enhances viral reactivation and latency.

Gammaherpesviruses are ubiquitous, lifelong pathogens associated with multiple cancers that infect over 95% of the adult population. Increases in viral reactivation, due to stress and other unknown factors impacting the immune response, frequently precedes lymphomagenesis. One potential stressor that could promote viral reactivation and increase viral latency would be the myriad of infections from bacterial and viral pathogens that we experience throughout our lives. Using murine gammaherpesvirus 68 (MHV68), a mouse model of gammaherpesvirus infection, we examined the impact of bacterial challenge on gammaherpesvirus infection. We challenged MHV68 infected mice during the establishment of latency with nontypeable Haemophilus influenzae (NTHi) to determine the impact of bacterial infection on viral reactivation and latency. Mice infected with MHV68 and then challenged with NTHi, saw increases in viral reactivation and viral latency. These data support the hypothesis that bacterial challenge can promote gammaherpesvirus reactivation and latency establishment, with possible consequences for viral lymphomagenesis.