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Objective To measure the expressions of Kaposi′s sarcoma-associated herpesvirus type 8 associated-microRNAs k12-1 (kshv-miR-k12-1)and k12-12 (kshv-miR-k12-12)in Kaposi′s sarcoma tissue, and to assess their relationship with pathological stage and lesion area of Kaposi′s sarcoma, HIV infection, and human herpesvirus type 8 (HPV-8)infection. Methods Totally, 18 paired tissue specimens stored in liquid nitrogen from Kaposi′ s sarcoma lesions and paralesional skin were collected. Total RNAs were extracted from these specimens by using Trizol reagent, and reversely transcribed into cDNA. SYBR Green real-time fluorescence-based quantitative PCR was performed to measure the expressions of kshv-miR-k12-1 and kshv-miR-k12-12 in these specimens. The relationship of kshv-miR-k12-1 and kshv-miR-k12-12 expressions with the pathological stage and lesion area of Kaposi′s sarcoma, HIV and HPV-8 infections was analyzed. Results Compared with paralesional normal skin, Kaposi′s sarcoma lesions showed significantly increased expressions of kshv-miR-k12-1 (2-ΔΔCt: 1.016 ± 1.645 vs. 0.029 ± 0.019, t = 2.542, P = 0.016)and kshv-miR-k12-12 (2-ΔΔCt: 2.104 ± 1.973 vs. 0.102 ± 0.093, t = 4.301, P = 0.000). There were no significant differences in the expressions of kshv-miR-k12-1 or kshv-miR-k12-12 between patients with HIV or HPV-8 infection and those without, among patients with different pathological stages of Kaposi′s sarcoma, or among patients with different lesion areas (all P > 0.05). Conclusion Both kshv-miR-k12-1 and kshv-miR-k12-12 are highly expressed in Kaposi′s sarcoma, but neither of their expressions is related to HIV or HPV-8 infection, pathological stage or lesion area of Kaposi′s sarcoma.
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Objective To investigate whether HIV-1 Nef could promote the angiogenesis and tu-morigenesis induced by KSHV vIL-6 through regulating PTEN/PI3K signaling pathway .Methods Lipo-some transfection was used to transfect cDNA of pPTEN , dominant-negative ( DN) construct of PI3K and control vector into endothelial cells , which stably express KSHV vIL-6 and HIV-1 Nef.Microtubule forma-tion assay and chicken chorioallantoic membrane ( CAM) assay were used to evaluate microtubule formation and angiogenesis , respectively .Expressions of PTEN and PI 3 K were measured by Western blot .Results Both overexpression of PTEN and inhibited expression of PI 3K suppressed the vIL-6-induced microtubule for-mation and angiogenesis in CAM mediated by Nef .Conclusion HIV-1 Nef enhances vIL-6-induced angio-genesis and tumorigenesis through regulating PTEN/PI3K signaling pathway .
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Kaposi's sarcoma associated herpesvirus (KSHV) is subdivided into gamma-herpesvirus and causes Kaposi's sarcoma in human immunodeficiency virus (HIV)-infected patients. A defining feature of herpesviral biology is the presence of two alternative genetic lifestyles - a latent infection and a lytic replicative cycle. Almost all herpesviruses examined so far have been shown to express viral miRNAs in latently and/or productively infected cells. KSHV encodes an array of 15 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. The expression of an array of these viral miRNAs in KSHV-infected cells suggests that down-regulation of host cell mRNAs by miRNA-mediated RNA interference may represent a critical step in the establishment and/or maintenance of latent infections by KSHV. To investigate KSHV miRNAs that are expressed in KSHV-infected cells, KSHV-infected human umbilical cord vein endothelial cells (HUVECs) and BCBL-1 cells were used and their miRNAs were analyzed by a modified real-time PCR method. Some KSHV miRNAs were detected in KSHV-infected HUVECs and their expression was affected by genetic life cycles. In addition, KSHV miRNAs were also detected in BCBL-1 and their expression was not related to treatment of sodium butyrate. These results indicate that KSHV infection in cells inducing KSHV miRNAs expression would be increased upon entry into latent replication.
Subject(s)
Humans , Biology , Butyrates , Down-Regulation , Endothelial Cells , Herpesviridae , Herpesvirus 8, Human , HIV , Life Cycle Stages , Life Style , MicroRNAs , Real-Time Polymerase Chain Reaction , RNA Interference , RNA, Messenger , Sarcoma, Kaposi , Sodium , Umbilical Cord , VeinsABSTRACT
Objective To package the recombinant lentivirus containing HIV-1 Vpr gene and detect the effect of Vpr protein expression on the latent infection and lyric replication of KSHV.Methods The fragment of Vpr gene from expression plasmid pCI-neo-Vpr was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant plasmid pHAGE-Vpr,package vector psPAX2 and envelope vector pMD2.G were cotransfected into 293T cells.GFP expression was observed by fluorescent microscopy.Culture media of 293T cells were harvested and filtered through 0.45 μm filter.After 293T cells were infected by a series of diluted lentivirus,the virus titer was checked by observing GFP expression.Vpr mRNA transcripts in 293T cells were detected by RT-PCR.Then BCBL-1 cells were infected by the recombinant lentivirus with 1 MOI,GFP expression was observed by fluorescent microscopy,and the mRNA transcripts and protein expression of Vpr in BCBL-1 cells were detected by RT-PCR and Western blot.Meanwhile,the mRNA transcripts and protein expression of KSHV lytic cycle gene Rta were detected by RT-PCR and Western blot,respectively.Results The recombinant lentivirus carrying HIV-1 Vpr was packaged successfully with the virus titer of 4 × 107 TU/ml.After infected with lentivirus,BCBL-1 cells could express GFP,and the exact band of Vpr was detectable by RT-PCR and Western blot.Moreover,the expression of KSHV Rta mRNA and protein were downregulated by Vpr protein.Conclusion Overexpression of HIV-1 Vpr mediated by the recombinant lentivirus could inhibit KSHV lytic replication and enhance KSHV latent infection.
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Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS),primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Hart people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).
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The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.
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Objective To establish antigen capture ELISA methed to detect Kaposi's sarcoma-as-sociated herpesvirus(KSHV)antigen,and to evaluate its feasibility for clinical application.Methods The BALB/c mice and New Zealand white rabbits were injected with purified recombinant KSHV gpK8.1 proteins to prepare the monoclonal antibody(McAb)and polyclonal antibody(PcAb)anti-gpK8.1,respectively.A new antigen capture ELISA method was established for KSHV antigen detection.The detection reproducibili-ty as well as the sensitivity and specificity of this new assay were determined by the optimization test,which antibody pairs were analyzed to choose the best coating antibody and detecting antibody.The 3 KSHV posi-tive patients sera and 257 patients sera from sexually transmitted disease,cancers or gynecological diseases were detected with this assay to evaluate its value for clinical application.Results When the McAb as coat-ing antibody at concentration of 5 μg/ml and PcAb as detecting antibody at concentration of 1.6μg/ml were selected,the highest P/N value could be obtained.The sensitive analysis of this test could detect recombi-nant KSHV gpK8.1 antigen of 31.28 ng/ml.Meanwhile,it is highly specific to detect KSHV antigen with-out cross reaction to Epstein-Barr vims(EBV),herpes simplex virus(HSV)-1 or HSV-2.All of three KSHV-positive sera and 4 sera from 257 clinical samples were positive with this new assay.which indicated that it could be used for capturing KSHV antigen.Conclusion A sensitive and specific McAb-based anti-gen capture ELISA method to detect KSHV antigen were established successfully.It is of great potential val-ue to develop reagent for KSHV clinical serologic dingnosis.
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To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E. coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.
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Tumor angiogenesis is the uncontrolled growth of blood vessels in tumors,serving to supply nutrients and oxygen,and remove metabolic wastes.Kaposi's sarcoma (KS),a multifocal angioproliferative disorder characterized by spindle cell proliferation,neo-angiogenesis,inflammation,and edema,is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV).Recent studies indicate that KSHV infection directly promotes angiogenesis and inflammation through an autocrine and paracrine mechanism by inducing pro-angiogenic and pro-inflammatory cytokines.Many of these cytokines are also expressed in KS lesions,implicating a direct role of KSI-IV in the pathogenesis of this malignancy.Several KSHV genes are involved in KSHV-induced angiogenesis.These studies have provided insights into the pathogenesis of KS,and identified potential therapeutic targets for this malignancy.
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Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS)patient.In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies:primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient's immune system in favor of tumor growth.As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor.Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.
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s Kaposi's sarcoma-associated herpesvirus (KSHV) was first identified as the etiologic agent of Kaposi's sarcoma (KS) in 1994.KSHV infection is necessary,but not sufficient for the development of Kaposi sarcoma (KS),primary effusion lymphoma (PEL),and multicentric Castleman disease (MCD).Advances in the prevention and treatment of KSHV-associated Diseases have been achieved,even though current treatment options are ineffective,or toxic to many affected persons.The identification of new targets for potential future therapies and the randomized trial to evaluate the efficacy of new antivirals are required.
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Kaposi's sarcoma-associated herpesvirus (KSHV) is the primary etiological agent of Kaposi's sarcoma, primary effusion lymphoma and muticentric Castleman's disease. In common with the other herpesviruses, KSHV exhibits both latent and lytic life cycles, both of which are characterized by distinct gene expression profiles and programs. KSHV encodes proteins which play essential roles in the inhibition of host adaptive and innate immunity, the inhibition of apoptosis, and the regulation of the cell cycle. KSHV also encodes several proteins which have transforming and intrcellular signalling activity.
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A putative gamma herpesvirus, termed human herpesvirus 8 (HHV-8), discovered in recent years, has been implicated as a possible etiologic agent for Kaposi`s sarcoma (KS). In South Korea, the incidence of KS in HIV seropositive individuals is very low. The cause of its rarity as compared with other countries is unclear. The objective of this study was performed to determine the prevalence of infection with HHV-8 and to clarify the cause of low incidence of KS in Korean populations including HIV seropositive individuals. The study population was composed of 200 blood donors, 220 voluntary visitors for sexual transmitted infection (STI)-testing in the public health centers, and 214 HIV-seropositive individuals. For the detection of HHV-8 antibodies, all blood samples were tested using Advanced Biotechnologies Inc`s enzyme-linked immunosorbent assay (ELISA) kits and the reactive samples were retested using Biotrin International SARL`s immunofluorescent assay (IFA). Also, we investigated the seroprevalence of Cytomegalovirus (CMV), Varicella-Zoster virus (VZV) and Epstein-Barr Virus (EBV) in order to get more information of HHV-8 and other human herpesviruses transmission in Korea. The prevalence of specific IgG to HHV-8 among HIV seropositive individuals was 7.0% {95% confidential interval: 4.0-11.3%}. The specific antibody to HHV-8 could be detected only in HIV seropositive men. The prevalences of antibodies to other human herpesviruses unlike HHV-8 were very high even in blood donors. These observations strongly suggest that the rarity of KS in this country may be caused by very low prevalence of HHV-8.
Subject(s)
Humans , Male , Antibodies , Biotechnology , Blood Donors , Cytomegalovirus , Enzyme-Linked Immunosorbent Assay , Herpesviridae , Herpesvirus 3, Human , Herpesvirus 4, Human , Herpesvirus 8, Human , HIV , Immunoglobulin G , Incidence , Korea , Prevalence , Public Health , Sarcoma , Sarcoma, Kaposi , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) been shown to be associated with human diseases including Kaposi's sarcoma, pleural effusion lymphoma, multicentric Castleman's disease. The IL-6 may both stimulate myeloma growth and prevent apoptosis of malignant plasma cells. Interestingly, viral IL-6(vIL-6), homolog to human interleukin-6(IL-6) in KSHV genome retains biologic activity. Thus, oncogenic role of the KSHV has been proposed as a pathogenesis of the multiple myeloma. We used ISH to determine the frequency of patients with multiple myeloma and plasmacytosis associated with KSHV-infected BM cells in fresh core biopsies and to determine the correlation between KSHV infection and clinical characteristics. METHODS: Bone marrow(BM) biopsy samples from 16 cases of multiple myeloma, 2 cases of monoclonal gammopathy of undetermined significance(MGUS) were obtained from the pathology division of Soon Chun Hyang University Hospital, Seoul, Korea. Biopsy sample of Kaposi's sarcoma for positive control and BM biopsy samples of myelodysplastic syndrome(MDS) and malignant lymphoma for negative control were obtained. Bitinylated probe to KSHV were prepared with the following sequences: 5' to 3' TGCAGCAGCTGTTGGTGTACCACATATCT. and in situ hybridization (ISH) was performed. RESULTS: Among the 18 patients. Two patients were MGUS and among 16 patients with multiple myeloma, 1 in stage IB disease, 1 stage IIB disease, 8 stage IIIA disease, 4 stage IIIB diseases and 2 in variant of multiple myeloma, extramedullary plasmacytoma. Strong positive signal was detected in nuclei and cytoplasm of the malignant cells of biopsy sample from 1 cases of Kaposi's sarcoma by ISH(positive control). Signal was not detected in BM biopsy samples of 7 cases from MDS and malignant lymphoma(negative control). Among 16 patients with multiple myeloma, 15 demonstrated viral positive cells and 2 cases with MGUS also showed viral positive cells by ISH. Signal was detected in nuclei and cytoplasm of stromal cells. Signal was strongly detected in MGUS than multiple myeloma. Positivity of the KSHV was not related with stage of the patients with multiple myeloma. One patients with multiple myeloma was studied at diagnosis and after chemotherapy. After chemotherapy KSHV was not detected. CONCLUSION: In MGUS and multiple myeloma, KSHV infects the stromal cells of BM rather than malignant plasma cells. On the basis of these data, we have supposed KSHV to play a role in transformation from MGUS to multiple myeloma. Particularly, due to the fact that signal of ISH was strongly detected in MGUS and was not detected in one case with multiple myeloma, it was presumed that KSHV was not major role in already advanced multiple myeloma but statistic significance was not demonstrated because of small numbers of cases. Further studies to reveal the correlation of KSHV and pathogenesis of multiple myeloma are needed.