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Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

Abstract Primary effusion lymphoma (PEL) and a form of multicentric Castleman’s disease (MCD) are both caused by Kaposi sarcoma herpesvirus (KSHV). There is a critical need for improved therapies for these disorders. The IL-6/JAK/STAT3 pathway plays an important role in the pathogenesis of both PEL and KSHV-MCD. We explored the potential of JAK inhibitors for use in PEL and KSHV-MCD, and found that pacritinib was superior to others in inhibiting the growth of PEL cell lines. Pacritinib induced apoptosis in PEL cells and inhibited STAT3 and NF-κB activity as evidenced by reduced amount of phosphorylated moieties. Pacritinib also inhibits FLT3, IRAK1, and ROS1; studies utilizing other inhibitors of these targets revealed that only FLT3 inhibitors exhibited similar cell growth inhibitory effects. FLT3’s likely contribution to pacritinib’s cell growth inhibition was further demonstrated by siRNA knockdown of FLT3. RNA sequencing and RT-PCR showed that many key host genes including cyclins and IL-6 were downregulated by pacritinib, while KSHV genes were variably altered. Finally, pacritinib suppressed KSHV viral IL-6-induced human IL-6 and IL-10 production in peripheral blood mononuclear cells, which may model an important step in KSHV-MCD pathogenesis. These results suggest that pacritinib warrants testing for the treatment of KSHV-MCD and PEL.

Abstract Background Kaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases. Methods RNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome. Results The transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4. Conclusions This study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm.

Abstract Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/β in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom’s effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

ABSTRACTPrimary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10–22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara’s effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Abstract Background The two oncogenic human gammaherpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein–Barr virus (EBV), both downregulate immune surface molecules, such as MHC-I, ICAM-1, and B7-2, enabling them to evade T-cell and natural killer cell immunity. Both also either encode for human cyclin homologues or promote cellular cyclin activity, and this has been shown to be important for proliferation and survival of gammaherpesvirus-induced tumors. CDK4/6 inhibitors, which are approved for certain breast cancers, have been shown to enhance expression of MHC-I in cell lines and murine models of breast cancer, and this was attributed to activation of interferons by endogenous retrovirus elements. However, it was not known if this would occur in gammaherpesvirus-induced tumors in which interferons are already activated. Methods Multiple KSHV/EBV-infected cell lines were treated with CDK4/6 inhibitors. The growth of viable cells and expression of surface markers was assessed. T cell activation stimulated by the treated cells was assayed by a T-cell activation bioassay. Both viral and host gene expression was surveyed using RT-qPCR. Results Three CDK4/6 inhibitors, abemaciclib, palbociclib, and ribociclib, inhibited cell growth in KSHV-induced primary effusion lymphoma (PEL) and EBV positive Burkitt’s lymphoma (BL) cell lines, and KSHV-infected human umbilical vein endothelial cells (HUVECs). Moreover, CDK4/6 inhibitors increased mRNA and surface expression of MHC-I in all three and prevented downregulation of MHC-I surface expression during lytic replication in KSHV-infected cells. CDK4/6 inhibitors also variably increased mRNA and surface expression of ICAM-1 and B7-2 in the tested lines. Abemaciclib also significantly enhanced T-cell activation induced by treated PEL and BL cells. Certain gammaherpesvirus genes as well as endogenous retrovirus (ERV) 3–1 genes were enhanced by CDK4/6 inhibitors in most PEL and BL lines and this enhancement was associated with expression of gamma interferon-induced genes including MHC-I. Conclusions These observations provide evidence that CDK4/6 inhibitors can induce expression of surface immune markers MHC-I, B7-2, and ICAM-1 in gammaherpesvirus-infected cell lines and induce virus-specific immunity. They can thus thwart virus-induced immune evasion. These effects, along with their direct effects on KSHV- or EBV-induced tumors, provide a rational for the clinical testing of these drugs in these tumors.

Background The Food and Drug Administration recommends that people living with HIV (PWH) with a CD4+ T cell count (CD4) ≥350 cells/µL may be eligible for any cancer clinical trial, but there is reluctance to enter patients with lower CD4 counts into cancer studies, including immune checkpoint inhibitor (ICI) studies. Patients with relapsed or refractory cancers may have low CD4 due to prior cancer therapies, irrespective of HIV status. It is unclear how baseline CD4 prior to ICI impacts the proportion of treatment-emergent adverse events (TEAE) and whether it differs by HIV status in ICI treated patients.Methods We conducted a pilot retrospective cohort study of participants eligible for ICI for advanced cancers from three phase 1/2 trials in the USA and Spain. We determined whether baseline CD4 counts differed by HIV status and whether the effect of CD4 counts on incidence of TEAE was modified by HIV status using a multivariable logistic regression model.Results Of 122 participants, 66 (54%) were PWH who received either pembrolizumab or durvalumab and 56 (46%) were HIV-negative who received bintrafusp alfa. Median CD4 at baseline was 320 cells/µL (IQR 210–495) among PWH and 356 cells/µL (IQR 260–470) among HIV-negative participants (p=0.5). Grade 3 or worse TEAE were recorded among 7/66 (11%) PWH compared with 7/56 (13%) among HIV-negative participants. When adjusted for prior therapies, age, sex, and race, the effect of baseline CD4 on incidence of TEAE was not modified by HIV status for any TEAE (interaction term p=0.7), or any grade ≥3 TEAE (interaction term p=0.1).Conclusions There was no significant difference in baseline CD4 or the proportions of any TEAE and grade ≥3 TEAE by HIV status. CD4 count thresholds for cancer clinical trials should be carefully reviewed to avoid unnecessarily excluding patients with HIV and cancer.

Here, using in vitro assays and structural analysis, the authors characterize the anti-SARS-CoV-2 properties of two small molcules, showing these to bind and target the virus main protease (Mpro), and to exhibit a synergistic antiviral effect when combined with remdesivir in vitro.

Human T cell leukemia virus type 1 (HTLV-1) persists in the host despite a vigorous immune response that includes cytotoxic T cells (CTL) and natural killer (NK) cells, suggesting the virus has developed effective mechanisms to counteract host immune surveillance. We recently showed that in vitro treatment of HTLV-1-infected cells with the drug pomalidomide (Pom) increases surface expression of MHC-I, ICAM-1, and B7-2, and significantly increases the susceptibility of HTLV-1-infected cells to NK and CTL killing, which is dependent on viral orf-I expression. We reasoned that by restoring cell surface expression of these molecules, Pom treatment has the potential to reduce virus burden by rendering infected cells susceptible to NK and CTL killing. We used the rhesus macaque model to determine if Pom treatment of infected individuals activates the host immune system and allows recognition and clearance of HTLV-1-infected cells. We administered Pom (0.2 mg/kg) orally to four HTLV-1-infected macaques over a 24 day period and collected blood, urine, and bone marrow samples throughout the study. Pom treatment caused immune activation in all four animals and a marked increase in proliferating CD4+, CD8+, and NK cells as measured by Ki-67+ cells. Activation markers HLA-DR, CD11b, and CD69 also increased during treatment. While we detected an increased frequency of cells with a memory CD8+ phenotype, we also found an increased frequency of cells with a Treg-like phenotype. Concomitant with immune activation, the frequency of detection of viral DNA and the HTLV-1-specific humoral response increased as well. In 3 of 4 animals, Pom treatment resulted in increased antibodies to HTLV-1 antigens as measured by western blot and p24Gag ELISA. Consistent with Pom inducing immune and HTLV-1 activation, we measured elevated leukotrienes LTB4 and LTE4 in the urine of all animals. Despite an increase in plasma LTB4, no significant changes in plasma cytokine/chemokine levels were detected. In all cases, however, cellular populations, LTB4, and LTE4 decreased to baseline or lower levels 2 weeks after cessation of treatment. These results indicated that Pom treatment induces a transient HTLV-1-specific immune activation in infected individuals, but also suggest Pom may not be effective as a single-agent therapeutic.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Multiple herpesviruses have been recently found to encode viral circular RNAs. Like cellular circular RNAs, these RNAs lack poly-A tails and their 5′ and 3′ ends have been joined, which confers protection from RNA exonucleases. We examined the expression patterns of circular RNAs from Kaposi’s sarcoma herpesvirus (KSHV) in various environments. We performed deep sequencing of circRNA-enriched total RNA from a KSHV-positive patient lymph node for comparison with previous circRNA-Seq results. We found that circvIRF4 is highly expressed in the KSHV-positive patient sample relative to both B cell lines and de novo infected primary vascular and lymphatic endothelial cells (LECs). Overall, this patient sample showed a viral circRNA expression pattern more similar to the pattern from B cell lines, but we also discovered new back-spliced junctions and additional viral circular RNAs in this patient sample. We validated some of these back-spliced junctions as circular RNAs with standard assays. Differential expression patterns of circular RNAs in different cell types led us to investigate what cellular factors might be influencing the ratio of viral linear mRNAs to circular RNAs. We found that repression of certain RNA-binding proteins shifted the balance between viral linear mRNAs and circular RNAs. Taken together, examining viral circular RNA expression patterns may become useful tools for discovering their functions, the regulators of their expression, and determining the stage and cell types of infection in humans.

Background Non-Hodgkin’s lymphoma (NHL) is currently the most common malignancy among people living with HIV (PLWH) in the USA. NHL in PLWH is more frequently associated with oncogenic viruses than NHL in immunocompetent individuals and is generally associated with increased PD-1 expression and T cell exhaustion. An effective immune-based second-line approach that is less immunosuppressive than chemotherapy may decrease infection risk, improve immune control of oncogenic viruses, and ultimately allow for better lymphoma control.Methods We conducted a retrospective study of patients with HIV-associated lymphomas treated with pembrolizumab±pomalidomide in the HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute.Results We identified 10 patients with stage IV relapsed and/or primary refractory HIV-associated NHL who were treated with pembrolizumab, an immune checkpoint inihibitor, with or without pomalidomide. Five patients had primary effusion lymphoma (PEL): one had germinal center B cell-like (GCB) diffuse large B cell lymphoma (DLBCL); two had non-GCB DLBCL; one had aggressive B cell lymphoma, not otherwise specified; and one had plasmablastic lymphoma. Six patients received pembrolizumab alone at 200 mg intravenously every 3 weeks, three received pembrolizumab 200 mg intravenously every 4 weeks plus pomalidomide 4 mg orally every day for days 1–21 of a 28-day cycle; and one sequentially received pembrolizumab alone and then pomalidomide alone. The response rate was 50% with particular benefit in gammaherpesvirus-associated tumors. The progression-free survival was 4.1 months (95% CI: 1.3 to 12.4) and overall survival was 14.7 months (95% CI: 2.96 to not reached). Three patients with PEL had leptomeningeal disease: one had a complete response and the other two had long-term disease control. There were four immune-related adverse events (irAEs), all CTCAEv5 grade 2–3; three of the four patients were able to continue receiving pembrolizumab. No irAEs occurred in patients receiving the combination of pembrolizumab and pomalidomide.Conclusions Treatment of HIV-associated NHL with pembrolizumab with or without pomalidomide elicited responses in several subtypes of HIV-associated NHL. This approach is worth further study in PLWH and NHL.

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi's sarcoma, a tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom's effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom's interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.

ABSTRACT We assessed various newly generated compounds that target the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC50] = 2.8 μM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography–mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds. IMPORTANCE Targeting the main protease (Mpro) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is necessary for the development of Kaposi's sarcoma (KS), which most often develops in HIV-infected individuals. KS frequently has oral manifestations and KSHV DNA can be detected in oral cells. Numerous types of cancer are associated with the alteration of microbiome including bacteria and virus. We hypothesize that oral bacterial microbiota affects or is affected by oral KS and the presence of oral cell-associated KSHV DNA. In this study, oral and blood specimens were collected from a cohort of HIV/KSHV-coinfected individuals all previously diagnosed with KS, and were classified as having oral KS with any oral cell-associated KSHV DNA status (O-KS, n = 9), no oral KS but with oral cell-associated KSHV DNA (O-KSHV, n = 10), or with neither oral KS nor oral cell-associated KSHV DNA (No KSHV, n = 10). We sequenced the hypervariable V1-V2 region of the 16S rRNA gene present in oral cell-associated DNA by next generation sequencing. The diversity, richness, relative abundance of operational taxonomic units (OTUs) and taxonomic composition of oral microbiota were analyzed and compared across the 3 studied groups. We found impoverishment of oral microbial diversity and enrichment of specific microbiota in O-KS individuals compared to O-KSHV or No KSHV individuals. These results suggest that HIV/KSHV coinfection and oral microbiota might impact one another and influence the development of oral KS.

Most chronic viruses evade T-cell and natural killer (NK) immunity through downregulation of immune surface markers. Previously we showed that Pomalidomide (Pom) increases surface expression of major histocompatibility complex class I (MHC-I) in Kaposi sarcoma-associated herpesvirus-infected latent and lytic cells and restores ICAM-1 and B7-2 in latent cells. We explored the ability of Pom to increase immune surface marker expression in cells infected by other chronic viruses, including human T-cell leukemia virus type-1 (HTLV-1), Epstein-Barr virus (EBV), human papilloma virus (HPV), Merkel cell polyoma virus (MCV), and human immunodeficiency virus type-1 (HIV-1). Pom increased MHC-1, ICAM-1, and B7-2/CD86 in immortalized T-cell lines productively infected with HTLV-1 and also significantly increased their susceptibility to NK cell-mediated cytotoxicity. Pom enhancement of MHC-I and ICAM-1 in primary cells infected with HTLV-1 was abrogated by knockout of HTLV-1 orf-1. Pom increased expression of ICAM-1, B7-2 and MHC class I polypeptide related sequence A (MICA) surface expression in the EBV-infected Daudi cells and increased their T-cell activation and susceptibility to NK cells. Moreover, Pom increased expression of certain of these surface markers on Akata, Raji, and EBV lymphoblastic cell lines. The increased expression of immune surface markers in these virus-infected lines was generally associated with a decrease in IRF4 expression. By contrast, Pom treatment of HPV, MCV and HIV-1 infected cells did not increase these immune surface markers. Pom and related drugs may be clinically beneficial for the treatment of HTLV-1 and EBV-induced tumors by rendering infected cells more susceptible to both innate and adaptive host immune responses.

ABSTRACT Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts. IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma with poor prognosis caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies have revealed that HIF-1α, which mediates much of the cellular response to hypoxia, plays an important role in life cycle of KSHV. KSHV infection promotes HIF-1α activity, and several KSHV genes are in turn activated by HIF-1α. In this study, we investigated the effects of knocking down HIF-1α in PELs. We observed that HIF-1α knockdown in each of two PEL lines leads to a reduction in both aerobic and anaerobic glycolysis as well as lipid biogenesis, indicating that HIF-1α is necessary for maintaining a metabolic state optimal for growth of PEL. We also found that HIF-1α suppression leads to a substantial reduction in activation of lytic KSHV genes, not only in hypoxia but also in normoxia. Moreover, HIF-1α knockdown led to a decrease in the expression of various KSHV latent genes, including LANA, vCyclin, kaposin, and miRNAs, under both normoxic and hypoxic conditions. These observations provide evidence that HIF-1α plays an important role in PEL even in normoxia. Consistent with these findings, we observed a significant inhibition of growth of PEL in normoxia upon HIF-1α suppression achieved by either HIF-1α knockdown or treatment with PX-478, a small molecule inhibitor of HIF-1α. These results offer further evidence that HIF-1α plays a critical role in the pathogenesis of PEL, and that inhibition of HIF-1α can be a potential therapeutic strategy in this disease.

Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.

Kaposi's sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.

The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.

Diffuse multi-spectral imaging has been evaluated as a potential non-invasive marker of tumor response. Multi-spectral images of Kaposi sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through principal component analysis (PCA) of the data. These images were compared with clinical and pathological responses determined by conventional means. We demonstrate that cutaneous lesions have increased blood volume concentration and that changes in this parameter are a reliable indicator of treatment efficacy, differentiating responders and non-responders. Blood volume decreased by at least 20% in all lesions that responded by clinical criteria and increased in the two lesions that did not respond clinically. Responses as assessed by multi-spectral imaging also generally correlated with overall patient clinical response assessment, were often detectable earlier in the course of therapy, and are less subject to observer variability than conventional clinical assessment. Tissue oxygenation was more variable, with lesions often showing decreased oxygenation in the center surrounded by a zone of increased oxygenation. This technique could potentially be a clinically useful supplement to existing response assessment in KS, providing an early, quantitative, and non-invasive marker of treatment effect.

Soon after the discovery of Kaposi sarcoma (KS)-associated herpesvirus (KSHV), it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD) arising in patients infected with human immunodeficiency virus. It has subsequently been recognized that KSHV–MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV–MCD is dominated by systemic inflammatory symptoms including fevers, cachexia, and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV–MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6). Patients with KSHV–MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h) IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV–MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS) has been described. Its clinical manifestations resemble those of KSHV–MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV–MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, homolog of human interleukin-6 and IL-10 comparable to those seen in KSHV–MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms seen in some patients with severe KS or primary effusion lymphoma. Additional research is needed to better define the clinical spectrum of KICS and its relationship to KSHV–MCD. In additional, research is needed to better understand the pathogenesis and epidemiology of both KICS and KSHV–MCD, as well as the optimal therapy for both of these disorders.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with human prostate cancer and chronic fatigue syndrome. Since retroviruses cause various cancers, and XMRV replication might be facilitated by HIV-1 co-infection, we asked whether certain patients with HIV-associated lymphomas are infected with XMRV. Analysis of PMBCs and plasma from 26 patients failed to detect XMRV by PCR, ELISA, or Western blot, suggesting a lack of association between XMRV and AIDS-associated lymphomas.

BackgroundMaturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.Methodology/principal findingsTo determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.Conclusions/significanceWe developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.

Hypoxia inducible factors (HIFs) are the principal means by which cells upregulate genes in response to hypoxia and certain other stresses. There are two major HIFs, HIF-1 and HIF-2. We previously found that certain genes are preferentially activated by HIF-2. One was protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). PTPRZ1 is overexpressed in a number of tumors and has been implicated in glioblastoma pathogenesis.To understand the preferential activation of PTPRZ1 by HIF-2, we studied the PTPRZ1 promoter in HEK293T cells and Hep3B cells. Through deletion and mutational analysis, we identified the principal hypoxia response element. This element bound to both HIF-1 and HIF-2. We further identified a role for ELK1, an E26 transformation-specific (Ets) factor that can bind to HIF-2alpha but not HIF-1alpha, in the HIF-2 responsiveness. Knock-down experiments using siRNA to ELK1 decreased HIF-2 activation by over 50%. Also, a deletion mutation of one of the two Ets binding motifs located near the principal hypoxia response element similarly decreased activation of the PTPRZ1 promoter by HIF-2. Finally, chromatin immunoprecipitation assays showed binding of HIF and ELK1 to the PTPRZ1 promoter region.These results identify HIF-binding and Ets-binding motifs on the PTPRZ1 promoter and provide evidence that preferential activation of PTPRZ1 by HIF-2 results at least in part from cooperative binding of HIF-2 and ELK1 to nearby sites on the PTPRZ1 promoter region. These results may have implications in tumor pathogenesis and in understanding neurobiology, and may help inform the development of novel tumor therapy.