Characterization of the OSSN Microbiome in HIV-1 Infected Patients

Purpose: Ocular surface squamous neoplasia (OSSN) is a rare cancer previously seen in elderly men. In Botswana there is an increase in OSSN and pterygia among young HIV-1 infected patients. Factors that determine the course of this cancer have not been characterized. Recent studies identified HPV, EBV, KSHV, HSV-1/2, and CMV in patient samples. We now characterize the microbiome associated with the disease that may contribute to its course. Results: Pyrosequencing identified viruses, bacteria, fungus and parasites. Analysis of shotgun cloning sequences showed a majority of infectious agents identified by pyrosequencing. Conclusion: HIV patients with OSSN in Botswana are infected with a range of infectious agents which may represent a unique microbiome. The persistent expressions of gene products by these agents some of which are oncogenic are likely to contribute to the oncogenic process and suggest that treatment modalities of the cancer should involve the screening for endemic agents

Oncogenic viruses associated with vulva cancer in HIV-1 patients in Botswana

BACKGROUND: Oncoviruses such as HPV, KSHV, and EBV have been reported in patients with HIV infection and AIDS. How oncovirus-associated cancers rise in AIDS patients has not been fully established. The purpose of our study was to identify the viral agents in vulvar cancer and to assess their contribution to pathogenesis. METHOD: We retrospectively identified a total of 13 vulva tissue samples from HIV-1 positive and 9 vulvar samples from HIV-1 negative patients from the Botswana National Health Laboratory in Gaborone, Botswana, a Southern African country with a high incidence of HIV. We utilized PCR and IHC to identify HPV, EBV, KSHV, and JC virus in FFPE preserved tissue samples. RESULTS: Using the GP5(+)/GP6(+) primer set we detected several HPV types in tissue samples. EBV was detected in all of the positive cases (100%) and in most of the negative cases (89%). KSHV was detected in 39% of the HIV-1 positive samples and in 11% of the negative samples, and no JC virus was detected in any of the samples. Using IHC we demonstrated that LANA was expressed in 61% of the positive samples and in 44% of the negative samples. The ubiquitous EBV was more consistently expressed in negative cases (100%) than in positive cases (69%). Interestingly, the HPV-16 E6 transcript was detected in 56% of the negative samples compared to 31% of the positive samples. However, the cell cycle protein P21 used as a surrogate marker for HPV was detected in 77% of the positive samples and in 44% of the negative samples, while VEGF signals were similar in both positive (92%) and negative samples (89%). CONCLUSION: Our study, suggests that in Botswana, vulvar squamous cell carcinoma (VSCC) is associated with oncogenic viruses present in the niche but the contribution and progression may be regulated by HPV and other immunosuppressive infections that include HIV-1

EC(5)S Ubiquitin Complex Is Recruited by KSHV Latent Antigen LANA for Degradation of the VHL and p53 Tumor Suppressors

Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposi's sarcoma–associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC(5)S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5–Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference–mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells

Hypoxia Inactivates the VHL Tumor Suppressor through PIASy-Mediated SUMO Modification

The hypoxic microenvironment contributes to embryonic development and tumor progression through stabilization of the potent transcriptional factor HIFα. In normoxia, the tumor suppressor protein VHL acts as an E3 ubiquitin ligase to target HIFα for proteolytic destruction. Increasing evidence shows that VHL is a multifunctional adaptor involved in inhibition of HIFα-dependent and independent cellular processes. However, the molecular effect of hypoxic stress on VHL functions remains elusive. Here we report that PIASy, a SUMO E3 ligase upregulated in hypoxia, interacts with VHL and induces VHL SUMOylation on lysine residue 171. Moreover, PIASy-mediated SUMO1 modification induces VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration and clonogenicity. Knockdown of PIASy by small interfering RNA leads to reduction of VHL oligomerization and increases HIF1α degradation. These findings reveal a unique molecular strategy for inactivation of VHL under hypoxic stress

Cytokine and Chemokine Expression Profiles in HIV-1 Infected Patients With Ocular Surface Squamous Neoplasia From Botswana

Purpose: Ocular surface squamous neoplasia (OSSN) rate has increased in incidence with the HIV pandemic in Africa. Multiple factors including cellular and environmental can affect the pathogenesis of OSSN in HIV-infected patients. We will investigate anti-inflammatory cytokines, proinflammatory cytokines, and growth factor expression in sera and tissue samples of OSSN and pterygia for the potential link to the development of OSSN. Results: Antibody analysis showed significant changes in levels of pro-inflammatory cytokines, anti-inflammatory cytokines and growth factors in sera. Quantitative RT-PCR of tissues showed expression of inflammatory cytokines and chemokines associated with HIV infection and carcinogenesis. Conclusion: Our findings showed that dysregulation in expression of cytokines and growth factors in patients with multiple infections may contribute to pathogenesis of OSSN and pterygia. The data reinforces the significance of in depth analysis of immune function in HIV-1 OSSN patients with multiple viral infections that has potential for therapy and vaccine development

Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis

The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities

Nm23-H1 can induce cell cycle arrest and apoptosis in B cells

Nm23-H1 is a well-known tumor metastasis suppressor, which functions as a nucleoside-diphosphate kinase converting nucleoside diphosphates to nucleoside triphosphates with an expense of ATP. It regulates a variety of cellular activities, including proliferation, development, migration and differentiation known to be modulated by a series of complex signaling pathway. Few studies have addressed the mechanistic action of Nm23-H1 in the context of these cellular processes. To determine the downstream pathways modulated by Nm23-H1, we expressed Nm23-H1 in a Burkitt lymphoma derived B-cell line BJAB and performed pathway specific microarray analysis. The genes with significant changes in expression patterns were clustered in groups which are responsible for regulating cell cycle, p53 activities and apoptosis. We found a general reduction of cell cycle regulatory proteins including cyclins and cyclin dependent kinase inhibitors (anti proliferation), and upregulation of apoptotic genes which included caspase 3, 9 and Bcl-x. Nm23-H1 was also found to upregulate p53 and downregulate p21 expression. A number of these genes were validated by real time PCR and results from promoter assays indicated that Nm23-H1 expression downregulated cyclin D1 in a dose responsive manner. Further, we show that Nm23-H1 forms a complex with the cellular transcription factor AP1 to modulate cyclin D1 expression levels. BJAB cells expressing Nm23-H1 showed reduced proliferation rate and were susceptible to increased apoptosis which may in part be due to a direct interaction between Nm23-H1 and p53. These results suggest that Nm23-H1 may have a role in the regulation of cell cycle and apoptosis in human B-cells

Kaposi's Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells

The early period of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs

Collision of Three Pandemics: The Coexistence of Cervical Cancer, HIV Infection, and Prior Tuberculosis in the Sub-Saharan Country of Botswana.

Cervical cancer is the leading cause of cancer-related mortality in the developing world, where HIV and Mycobacterium tuberculosis (TB) infection are also endemic. HIV infection is independently associated with increased morbidity and mortality among women with cervical cancer. TB is believed to increase the risk of malignancies and could cause chronic inflammation in the gynecologic tract. However, the relationship between cervical cancer and TB in settings hyperendemic for HIV is unknown. We found that 18 (10%) of a cohort of 180 women with cervical cancer in Botswana had a history of TB disease. Age and HIV infection were also associated with a history of TB disease. Our data show that prior TB disease is highly prevalent among patients with cervical cancer infected with HIV. The coexistence of cervical cancer, HIV infection, and prior TB infection might be higher than expected in the general population. Prospective studies are needed to better determine the impact of the collision of these three world health epidemics