Kaposi's Sarcoma Herpesvirus DNA Research Articles

Identification of allosteric regulation sites of Kaposis sarcoma virus DNA polymerase to discover its specific inhibitors via analysis of correlated intramolecular motions

Human herpesvirus 8 (HHV8) also known as Kaposi sarcoma herpesvirus (KSHV) is a DNA virus associated with several dangerous diseases (Castleman disease, inflammatory cytokine syndrome, Kaposi sarcoma itself, etc). The exceptional importance of DNA-dependent DNA polymerases (DNA pols) in double-strand DNA viruses' lifecycle makes them attractive targets for specific low-weight molecular inhibitors, which can be used as antiviral drugs. Among the drug discovery strategies, the search/design of allosteric inhibitors of target enzymes seems to be the most promising. As a rule, allosteric regulatory sites have individual amino acid compositions, and the selectivity of the appropriate allosteric inhibitors is much higher compared to competitive inhibitors, especially in the case of nucleotide-binding enzymes. The general structural regularities of the location of allosteric regulation sites on the protein surface remain unclear. However, it is quite natural to assume some interdependence between behavior of amino acids in active and allosteric binding site that can be revealed by correlation analysis of intramolecular motions. Thus, the aim of this work was identification of site(-s) of allosteric regulation of KSHV DNA pol for the next rational design / search for compounds - potential drugs against KSHV dependent diseases. According to the aim the following tasks are set: to calculate the molecular dynamics of KSHV DNA polymerase as well as HIV reverse transcriptase for which allosteric regulation sites are known; to evaluate a correlation between motions in active and allosteric site of HIV reverse transcriptase and on the basis of these data to determine a localization of such site(-s) on the surface of KSHV DNA polymerase; to carry out a molecular docking of several compounds with known antipolymerase activity into the identified site of allosteric regulation and estimate the appropriate binding energies. As a primary result, the correlation between the motions of amino acid residues in the active and allosteric site of HIV reverse transcriptase was revealed. The corresponding correlation coefficients are in the range of 0.55–0.62. Thus, the initial hypothesis about interdependence of the behavior of the active site and the site of allosteric regulation was confirmed. In the KSHV DNA pol was revealed a region in the range from 420 to 460 amino acid residue which motions correlate with the motions of the active site of this enzyme. The correlation coefficients are in the range of 0.52–0.57. Analysis of the molecular surface of the KSHV DNA pol revealed a site able to bind small organic compounds, which seems to be allosteric regulation site of KSHV DNA pol. As a result of molecular docking, two compounds with binding energies of -22.41 and -20.65 kJ/mol respectively were identified as potential non-competitive inhibitors of KSVH DNA pol. The identified compounds can be structural prototypes for the development of new drugs against KSHV caused pathologies. The proposed approach to the identification of allosteric regulatory sites by analysis of intramolecular correlated motions can be used for rational design/search of high selective inhibitors with a non-competitive mechanism of action for a wide range of enzymes.

IL-13 Expression Characterizes Effusions Associated with Primary Effusion Lymphoma but Not Other Disorders Caused By Kaposi Sarcoma Herpesvirus (KSHV)

BACKGROUND: Kaposi sarcoma herpesvirus (KSHV, also called human herpesvirus-8) is the causative agent of 3 disorders: primary effusion lymphoma (PEL), Kaposi sarcoma (KS), and a plasmablastic form of multicentric Castleman disease (KSHV-MCD). It also causes KSHV inflammatory cytokine syndrome (KICS), which is characterized by inflammatory symptoms and an elevated KSHV viral load. Multiple KSHV-associated diseases, which usually develop in HIV-infected patients, can present together in the same patient. Effusions can occur in each of these diseases, thereby presenting a diagnostic challenge. Identifying PEL is especially crucial as prompt treatment with multi-agent chemotherapy can be curative. We analyzed effusions from patients with KICS, PEL, and KSHV-MCD to identify distinct immunologic characteristics and virologic profiles that may aid diagnosis, inform treatment and elucidate pathogenesis.PATIENTS AND METHODS: We identified 22 HIV-infected patients with effusions [pleural effusions (20), ascites (1) and pericardial effusion (1)] with diagnoses of PEL (9 patients), KICS (8 patients), or KSHV-MCD (5 patients). All patients had a concurrent diagnosis of KS. We obtained clinical and immunologic characteristics from effusions and paired serum samples at the same timepoint for each patient. Serum and effusion cytokine levels of interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, and IL-12p70; interferon gamma (IFN-g); tumor necrosis factor alpha (TNF-a); vascular endothelial growth factor (VEGF); and inducible protein 10 (IP-10) were evaluated using a commercial multiplex assay. Peripheral blood mononuclear cell (PBMC) and effusion- associated KSHV and Epstein Barr virus (EBV) viral DNA (KSHV-VL, EBV-VL) in PBMC and cells were quantified using PCR with primers to KSHV K6 and EBV pol. Effusion and serum immunologic and virologic characteristics were compared within each disease and separately between diseases using the Wilcoxon sign rank test and Wilcoxon rank sum test, respectively. In these exploratory analyses with few patients, no correction was made for multiple comparisons.RESULTS: In patients with PEL, the median (med) age was 42 years with med CD4+ count of 54 T-cells/μL and HIV viral load (VL) of 325 copies/mL. In those with KICS, the med age was 32 years, with a med CD4+ count of 119 T-cells/μL and HIV VL of 48 copies/mL. In patients with KSHV-MCD, the med age was 31 years, med CD4+ count of 118 T-cells/μL and HIV VL was 75 copies/mL.IL-13 was substantially higher in PEL effusions as compared to serum levels (med 16.9 vs. <0.12 ng/ml; p=0.007). In addition, patients with PEL had significantly higher levels of 6 other cytokines (IL-12p70, IL-1ß, IL-2, IL-4, IL-6 and IP-10) in effusions as compared with serum (Table 1, p<0.05). In both KSHV-MCD and KICS, IL-12p70, IL-2 and IL-4 levels were higher in the effusion as compared with serum (p<0.05).In analyses comparing serum and cytokine differences among diseases, effusions from patients with PEL had detectable levels of IL-13 (med 16.9 ng/ml; interquartile range 9.7-26.9 ng/ml) compared to patients with KSHV-MCD (med <0.114 ng/ml; p=0.0037) or KICS (med <0.114 ng/ml; p=0.0003). PEL effusions had higher IL-1ß levels as compared with KICS effusions (p=0.0028). Serum IL-10 levels were also higher in PEL as compared with KICS (med 51.6 vs. 2.5ng/mL; p=0.0015). KSHV VL levels were significantly higher in PEL effusions as compared to KICS effusions (med 31,428,571 vs. 569 copies/mL; p=0.0005) and KSHV-MCD (med 231,884 copies/mL; p=0.02).CONCLUSIONS: In HIV-infected patients, effusions can indicate a diagnosis of PEL, KSHV-MCD and/or KICS. Quantifying KSHV VL in the effusion may be useful in the diagnosis of PEL. Effusions in PEL had a distinct profile compared to the circulation or other KSHV-associated diseases, particularly with regard to elevated IL-13, which may aid in diagnosis. In contrast, the inflammatory and virologic findings in KSHV-MCD and KICS effusions roughly paralleled the levels seen in the circulation. This study suggests that KSHV upregulation of IL-13 is a unique feature of PEL, which may contribute to PEL pathogenesis through STAT6 activation and be a potential future therapeutic target. [Display omitted] DisclosuresUldrick:Celgene: Patents & Royalties: 10,001,483 B2; Celgene: Research Funding; Merck: Research Funding. Yarchoan:Celgene Corp.: Research Funding; NIH: Patents & Royalties: Patents on IL-12 for KS and cereblon-binding drugs for KSHV diseases. Spouse has patent on KSHV IL-6. Patents assigned to DHHS/NIH..

Vascular endothelial growth factor A polymorphism and risk of Kaposi's sarcoma herpesvirus viremia in kidney allograft recipients.

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease in immunocompromised patients including allograft recipients. Detection of KSHV DNA in blood, as well as host genetic polymorphisms has been found to be associated with an increased risk for KS. We investigated an association between single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor A (VEGFA) gene region and KSHV viremia in kidney transplant recipients (KTR) in Saudi Arabia. In total, 152 KTR who have survived kidney transplantation for at least 6 months were included in the study. KSHV viremia was determined by real-time polymerase chain reaction (PCR). Genotyping of SNPs in the VEGFA region was performed by PCR and direct sequencing, as well as by restriction fragment length polymorphism. KSHV DNA was detected in 28.9% (n = 44) of the study population. The A-allele at position C172A VEGFA gene promoter region was found to be associated with KSHV viremia (odd ratio [OR] = 4.8, P = 0.005). In addition, the G-allele at position C+405G in the 5'-untranslated region was associated with KSHV viremia in women, but not in men (OR = 3.98, P = 0.004). Our results suggest an association of VEGFA polymorphisms with KSHV viremia among KTR in this study population. A limitation of our study is that the results can only be predicated for patients 6 months after kidney transplantation and should be validated in another cohort with larger sample size.

British HIV Association guidelines for HIV‐associated malignancies 2014

<scp>B</scp>ritish <scp>HIV</scp> Association guidelines for <scp>HIV</scp>‐associated malignancies 2014

Human Herpesvirus Replication and Abnormal CD8+ T Cell Activation and Low CD4+ T Cell Counts in Antiretroviral-Suppressed HIV-Infected Patients

BackgroundMost HIV-infected patients receiving virologically suppressive antiretroviral therapy continue to have abnormal, generalized T cell activation. We explored whether the degree of ongoing cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) replication was associated with higher virus-specific T cell activation and the failure to achieve normal absolute CD4+ T cell counts in the face of long-term suppressive antiretroviral therapy.MethodologyLongitudinally collected PBMC and saliva specimens obtained from HIV-infected patients on effective antiretroviral therapy for at least one year (plasma HIV RNA <75 copies/mL) were examined using a multiplex CMV, EBV and KSHV DNA PCR assay. Eleven cases were chosen who had CD8+ T cell CD38+HLA-DR+ expression >10% and plateau absolute CD4+ T cell counts <500 cells/µL. Five controls from the same study had CD8+ T cell CD38 expression <10% and plateau absolute CD4+ T cell counts >500 cells/µL.Results and ConclusionsAmong all subjects combined, 18% of PMBC samples were positive for CMV DNA, and 27%, 73% and 24% of saliva samples were positive for CMV, EBV and KSHV DNA, respectively. No significant differences or trends were observed between cases and controls in proportions of all CMV, EBV or KSHV DNA positive specimens, proportions of subjects in each group that intermittently or continuously shed CMV, EBV or KSHV DNA in saliva, or the median number of genome copies of CMV, EBV and KSHV DNA in saliva. Overall, number of genome copies in saliva were lower for KSHV than for CMV and lower for CMV than for EBV. Although replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients, we observed no evidence in this pilot case-control study that the magnitude of such human herpesvirus replication is associated with abnormally increased CD8+ T cell activation and sub-normal plateau absolute CD4+ T cell counts following virologically suppressive antiretroviral therapy.

Effects of Chemotherapy in AIDS-Associated Non-Hodgkin's Lymphoma on Kaposi's Sarcoma Herpesvirus DNA in Blood

To determine the relative frequency with which Kaposi's sarcoma-associated herpesvirus/HHV-8 (KSHV) DNA is detected in peripheral-blood mononuclear cells (PBMCs) and in plasma of patients with AIDS-KS and AIDS-associated non-Hodgkin's lymphoma (NHL; AIDS-NHL); to determine whether the presence of viral DNA in plasma reflects lysis of tumor cells or reflects the presence of viremia (ie, virion-encapsidated DNA); and to determine the effect of lymphoma therapy on KSHV DNA. Samples were obtained from patients enrolled in AIDS Malignancy Consortium clinical trials and from healthy donors. Real time PCR was used to quantify KSHV DNA in peripheral blood mononuclear cells (PBMC) and plasma. DNase digestion and fragment size determination studies were used to characterize the DNA detected. In patients with AIDS-KS, KSHV DNA was detected in PBMC (54%) and in plasma (62%). In patients with AIDS-NHL, KSHV DNA was detected in PBMC (19%) and in plasma (22%). Median copy numbers also differed. KSHV DNA in plasma appeared to be encapsidated. In six patients with AIDS-NHL who were treated with chemotherapy (with or without rituximab), KSHV copy number declined in PBMC and in plasma. KSHV DNA is sometimes detected in PBMC or in plasma of patients with AIDS-NHL without KS. Among patients with KSHV DNA detected in PBMC or in plasma, copy number does not distinguish between patients with AIDS-NHL and AIDS-KS. KSHV DNA in plasma likely reflects viremia and not simply lysis of tumor or other KSHV-infected cells. KSHV DNA copy number in PBMC and in plasma declined with lymphoma-directed cytotoxic chemotherapy in each of the six patients studied.

Kaposi Sarcoma Herpes Virus-associated Hemophagocytic Syndrome Complicated by Multicentric Castleman Disease and Kaposi Sarcoma in a HIV-negative Immunocompetent Patient: An Autopsy Case

Kaposi sarcoma herpes virus (KSHV), also known as human herpesvirus-8, plays an important role in the pathogenesis of Kaposi sarcoma (KS), multicentric Castleman disease (MCD) of the plasma cell type, and primary effusion lymphoma. KSHV is rarely associated with the hemophagocytic syndrome (HPS), but when it does occur, it most occurs in immunocompromised patients. We report herein an unusual case of KSHV-associated HPS in an immunocompetent patient. A previously healthy 62-yr-old male was referred for evaluation of leukocytopenia and multiple lymphadenopathies. After a lymph node biopsy, he was diagnosed with MCD of the plasma cell type. KSHV DNA was detected in the lymph node tissue by polymerase chain reaction. Following a short-term response of the leukocytopenia to prednisolone, mental change, left side weakness, fever, thrombocytopenia, hemolytic anemia, and renal failure developed. Despite intravenous immunoglobulin therapy and plasmapheresis, he expired. The lymph nodes were infiltrated by hemophagocytic histiocytes in the sinuses. Pulmonary nodules and gastric erosions were shown to be KS. KSHV DNA was detected in the stomach, lung, and liver. This is the first case of multiple KSHV associated diseases including MCD and KS with KSHV-associated hemophagocytic syndrome in an HIV-negative, non-transplant, immunocompetent patient.

KSHV/HHV8‐associated lymphomas

This review looks at the current state of knowledge on primary effusion lymphoma (PEL) and other Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus 8 (HHV8)-associated lymphomas. In 1995, KSHV DNA sequences were identified within a distinct subgroup of acquired immunodeficiency syndrome-related non-Hodgkin lymphomas localized in body cavities and presenting as pleural, peritoneal and pericardial lymphomatous effusions. Subsequently, the spectrum of KSHV/HHV8-associated lymphomas has been expanded by the identification of cases of extracavitary solid lymphomas without serous effusions. Despite the diversification in the clinical presentation of KSHV/HHV8-associated lymphomas, the majority of the cases reported demonstrated similar morphology, immunophenotype and KSHV/HHV8 viral status. KSHV/HHV8 infection is also in multicentric Castleman disease-associated plasmablastic lymphoma. The exact oncogenic mechanisms of KSHV/HHV8 are not clearly defined. The prognosis for KSHV/HHV8-associated lymphomas is poor. Novel approaches for therapy, outside traditional chemotherapy with CHOP (cyclophosphamide, doxorubicin, prednisone, vincristine), have been suggested. These include the addition of antiviral therapy as well as inhibition of specific cellular targets.

A new primary effusion lymphoma-derived cell line yields a highly infectious Kaposi's sarcoma herpesvirus-containing supernatant.

A primary effusion lymphoma (PEL) cell line, JSC-1, that yields highly infectious Kaposi's sarcoma herpesvirus (KSHV) supernatants was established from the ascitic fluid of a human immunodeficiency virus-positive patient. Flow cytometry showed strong expression of CD45 and lambda light-chain restriction. Southern blot hybridization showed immunoglobulin heavy-chain gene rearrangements in the tumor and the resultant cell line consistent with B-cell lineage. Expression of viral genes was assessed by reverse transcription-PCR and immunohistochemistry. Only latent Epstein-Barr virus (EBV) gene expression was detected, and this was at a low level. In contrast, lytic and latent KSHV gene expression were detected. Tetradecanoyl phorbol acetate and butyrate upregulated KSHV lytic expression, but not EBV lytic expression. Viral supernatant from JSC-1 was much more efficient at infecting primary human dermal microvascular endothelial cells (DMVECs) with KSHV than supernatants from BC-3 or BCP-1 PEL cell lines. Quantitation of viral yields produced by the PEL lines showed at least 2 orders of magnitude more DNase I-resistant KSHV DNA in the JSC-1 supernatant compared to BC-3 or BCP-1 supernatants. KSHV infection in DMVECs was associated with a change from a cobblestone to a spindle shape, LANA expression, and an increased number of mitoses.

Exacerbations of clinical symptoms in human immunodeficiency virus type 1-infected patients with multicentric Castleman's disease are associated with a high increase in Kaposi's sarcoma herpesvirus DNA load in peripheral blood mononuclear cells.

The epidemiologic link between multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS) and the high frequency of KS herpesvirus (KSHV) detection in both diseases raise the question of a role of this new virus in the pathogenesis of MCD. To explore this hypothesis, the KSHV DNA load was investigated in peripheral blood mononuclear cells of 3 human immunodeficiency virus (HIV)-infected patients with MCD at different points during the clinical course. Clinical parameters, such as fever and the presence of lymphadenopathy, were systematically assessed. Hemogram and C-reactive protein level determinations were performed as standard procedures. KSHV DNA load was investigated by means of semiquantitative polymerase chain reaction assay using peripheral blood mononuclear cells of the patients. A correlation between the variation in clinical and biologic parameters related to MCD and KSHV DNA load was found, suggesting a close relationship between KSHV and MCD in HIV-1-infected patients.

A PHASE II STUDY OF THALIDOMIDE FOR KAPOSI'S SARCOMA

BACKGROUND: Thalidomide may have three possible mechanisms of action against Kaposi's sarcoma (KS). It is a selective inhibitor of synthesis of TNFα (an autocrine growth factor in KS), has anti-angiogenic activity in vivo and reduces HIV-1 activation in peripheral blood mononuclear cells (PBMC) in vitro. There is one case report of activity of thalidomide in AIDS KS (Soler et al., 1996). The detection of KSHV (Kaposi's sarcoma herpes virus) in the PBMCs of HIV-seropositive patients by polymerase chain reaction (PCR) has been shown to correlate with the presence of KS and to predict for its development. METHODS: A single institution open label phase II study was designed to establish the activity and toxicity of thalidomide in the treatment of AIDS KS. KSHV DNA levels in PBMCs were determined by PCR quantification and correlated with clinical response. Response evaluation was performed using the AIDS Clinical Trials Group (ACTG) criteria. Only male patients were eligible in view of the teratogenicity of thalidomide. The treatment schedule was thalidomide 100mg orally once at night for an initial study period of two months. RESULTS: Seventeen patients median age 40 years (range 30-48) were enrolled and 11 are evaluable for response. The median CD4 cell count was 36 cells/µl(range 4-552) and 8/17 had prior AIDS defining opportunistic infections. Four patients developed a rash and one Raynaud's syndrome. Six patients were unevaluable due to early withdrawal (toxicity in four patients, progressive disease in one and poor compliance in one). Five of the 11 evaluable patients achieved a partial response. Thus the overall response rate is 45% (95% confidence interval 10-81%). A further two patients had stable disease (18%: 95% confidence interval 0-45%). Results of the PCR quantification for KSHV DNA will be presented. CONCLUSIONS: Although the number of patients in this study is small and therefore the confidence intervals wide, thalidomide appears to have activity in the treatment of AIDS related KS and deserves further investigation. Reference: Soler R, Howard M, Brink N, Gibb D, Tedder R & Nadal D. Regression of AIDS-related Kaposi's sarcoma during therapy with thalidomide. Clin. Infect. Dis 1996:23, 501-503. Acknowledgement: MB and KF are supported by a grant from the Crusaid and Star Foundation.

Kaposi's sarcoma-associated herpesvirus DNA sequences in AIDS-related and AIDS-unrelated lymphomatous effusions.

Primary effusions presenting as the sole lymphoma localization are also known as body-cavity-based-lymphoma (BCBL), and have been shown to carry Kaposi's sarcoma herpesvirus (KSHV) DNA sequences. The aim of this study was a comparative analysis of the clinical, pathologic and molecular features of BCBL and lymphomatous effusions secondary to tissue-based lymphomas occurring both in the general population and in HIV-1-infected individuals. All the lymphomatous effusion samples (seven AIDS-related and nine AIDS-unrelated) were subjected to an identical multiparameter investigation, including collection of clinical data, analysis of morphology and immunophenotype, as well as the study of viral sequences and genetic lesions. In six cases defined as BCBL (four AIDS-related and two AIDS-unrelated), the patients exhibited exclusive or predominant involvement of the body cavities. BCBL tended to display indeterminate phenotypes (4/6), whereas all AIDS-related and AIDS-unrelated lymphomatous effusions secondary to tissue-based lymphomas consistently expressed B-cell phenotype. Detection of KSHV DNA sequences was restricted to cases of BCBL (3/4 AIDS-related and 1/2 AIDS-unrelated), whereas EBV association (3/4) and expression of EBV-encoded antigens (LMP-1, 2/3; EBNA-2, 1/3) were confined to the AIDS-related BCBL. Overall, our results confirm that both AIDS-related and AIDS-unrelated BCBL preferentially associate with peculiar clinical, immunophenotypic and molecular features among lymphomatous effusions and therefore should be singled out as a specific clinico-pathologic entity.