Primary effusion lymphoma cells undergoing human herpesvirus type 8 productive infection produce C-type retroviral particles.

Primary effusion lymphomas (PELs) are invariably infected by the human herpesvirus 8 (HHV8)that is present in most PEL cells as latent virus but replicates in a subset of permissive cells to produce infectious progeny. Here we show that productively infected PEL cells release C-type retrovirus-like particles encoding an Mn++-dependent RT activity, which is typical of endogenous retroviruses. Strikingly, C-type particles are produced only in cells showing advanced HHV8 morphogenesis. Phorbol esters, which induce productive HHV8 replication and morphogenesis in PEL cells, increase RLP production. Phosphonoacetic acid, a blocker of HHV8 late gene expression, inhibits the production of C-type particles, whereas neutralizing anti-alphaIFN antibodies, which are known to increase HHV8 assembly, increases C-type particle production. These data suggest that factors expressed in advanced stages of HHV8 reactivation support endogenous C-type particle morphogenesis in PEL cells.

Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor.

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection.

Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.

BACKGROUND: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. METHODS: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. RESULTS AND CONCLUSIONS: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. IMPLICATIONS: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.

Human herpesvirus 8 seropositivity and risk of Kaposi's sarcoma and other acquired immunodeficiency syndrome-related diseases.

BACKGROUND: The incidence of Kaposi's sarcoma (KS) is increased severalfold in individuals infected with human immunodeficiency virus-1 (HIV). Human herpesvirus 8 (HHV8) has also been implicated in KS. We investigated several factors that may determine the onset of KS, particularly HHV8 infection in individuals after becoming seropositive for HIV. METHODS: We studied 366 individuals belonging to different HIV-exposure categories (i.e., homosexual activity, intravenous drug use, and heterosexual contact) for whom a negative HIV serologic test and then a positive HIV serologic test were available within a 2-year period. HHV8 antibody testing was performed by use of an immunofluorescence assay on the first serum sample available after the first positive HIV test. Actuarial rates of progression of KS and of other acquired immunodeficiency syndrome (AIDS)-defining diseases were estimated by use of time-to-event statistical methods. All statistical tests were two-sided. RESULTS: Twenty-one of the 366 study participants developed AIDS-related KS, and 83 developed AIDS without KS. One hundred forty (38.3%) participants had detectable anti-HHV8 antibodies. The actuarial progression rate to KS among persons co-infected with HIV/HHV8 was nearly 30% by 10 years after HIV seroconversion. Increasing HHV8 antibody titers increased the risk of developing KS (for seronegative versus highest titer [1:125 serum dilution], adjusted relative hazard [RH] = 51.82; 95% confidence interval [CI] = 6.08-441.33) but not of other AIDS-defining diseases (adjusted RH = 1.14; 95% CI = 0.72-1.80). HHV8-seropositive homosexual men compared with HHV8-seropositive participants from other HIV-exposure categories showed an increased risk of KS that approached statistical significance (adjusted RH = 6.93; 95% CI = 0.88-54.84). CONCLUSIONS: Approximately one third of individuals co-infected with HIV/HHV8 developed KS within 10 years after HIV seroconversion. Progression to KS increased with time after HIV seroconversion. Higher antibody titers to HHV8 appear to be related to faster progression to KS but not to other AIDS-defining diseases.

Reactivation and role of HHV-8 in Kaposi's sarcoma initiation.

Kaposi's sarcoma (KS) is an angioproliferative disease occurring in several clinical-epidemio-logic forms but all associated with infection by the human herpesvirus-8 (HHV-8). At least in early stages, KS is a reactive disease associated with a state of immune dysregulation characterized by CD8+ T-cell activation and production of Th1-type inflammatory cytokines (IC) that precedes lesion development. In fact, evidence indicates that IC can trigger lesion formation by inducing the activation of endothelial cells that leads to adhesion and tissue extravasation of lymphomonocytes, spindle cell formation, and angiogenesis, and HHV-8 reactivation that, in turn, leads to virus spread to all circulating cell types and virus dissemination into tissues. Due to virus escape mechanisms and deficient immune responses toward HHV-8, virus reactivation and spread are not controlled by the immune system but induce immune responses that may paradoxically exacerbate the reactive process. The virus is recruited into "activated" tissue sites where it finds an optimal environment for growth. In fact, viral load is very low in early lesions, whereas almost all spindle cells are infected in late-stage lesions. Although early KS is a reactive process of polyclonal nature that can regress, in time and in the presence of immunodeficiency, it can progress to a true sarcoma. This is likely due to the long-lasting expression of HHV-8 latency genes in spindle cells associated with the deregulated expression of oncogenes and oncosuppressor genes and, for AIDS-KS, with the effects of the HIV-1 Tat protein.

Human herpesvirus-8 and Kaposi's sarcoma: relationship with the multistep concept of tumorigenesis.

Kaposi's sarcoma (KS) develops through discrete inflammatory-angiogenic stages of polyclonal nature (early-stage lesions) to monomorphic nodules of spindle-shaped cells that can be clonal (late-stage lesions) and resemble true sarcomas. Molecular and epidemiological studies indicate that development of KS is tightly associated with infection by the human herpesvirus-8 (HHV-8). However, only individuals with specific conditions of immunodysregulation develop KS. In these individuals the systemic and tissue increase of Th-1-type cytokines (IC) reactivate HHV-8 infection, leading to increased viral load, antibody titers, and an expanded cell tropism that precedes the clinical appearance of KS. Recruitment of the virus into tissues by infected monocytes and other cell types is facilitated by the endothelial cell activation due to IC. In clinical lesions, HHV-8 infection increases with lesion stage and in late-stage lesions most of the spindle cells are latently infected, whereas only few lyrically infected cells are present, suggesting that latent genes may have a role in the transformation of the early inflammatory-hyperplastic lesion into a real sarcoma. The development of tumors, however, is regulated through a multistep process based on the acquisition by cells of several different capabilities leading to malignant growth. Here we review the available data on the expression of HHV-8-encoded genes in primary KS lesions and, in view of their biological activity, analyze their potential function in different steps of tumorigenesis. By this pragmatic approach interesting insights into potential key functions of HHV-8-encoded genes are found and steps of potential cooperativity with other viral factors (HIV-1-Tat) in the pathogenesis of KS are identified.

Reciprocal in vitro interactions between human herpesvirus-6 and HIV-1 Tat.

The transactivation induced by human herpesvirus 6 (HHV-6) on the HIV-1 promoter was studied both in the presence and in the absence of the retroviral transactivator protein (Tat) constitutively expressed in Jurkat cells. Jurkat-tat cells were infected with HHV-6, transfected with a plasmid containing HIV-1 long terminal repeat (LTR)/chloramphenicol acetyltransferase (CAT), and CAT assays were performed. HHV-6 infection in the presence of Tat resulted in significantly higher LTR activation than that observed by Tat or HHV-6 alone, indicating that HHV-6 and Tat interact synergically. Furthermore, it was demonstrated that expression of HIV-1 tat inhibits HHV-6 replication, as shown by a 3.6-15.4-fold reduction in infectious yield. We suggest that HHV-6 could trigger HIV reactivation in HIV-seropositive patients which, in turn, could inhibit HHV-6 production.

Incidence of Kaposi's sarcoma and HHV-8 seroprevalence among homosexual men with known dates of HIV seroconversion. Italian Seroconversion Study.

OBJECTIVES: To evaluate temporal trends of Kaposi's sarcoma (KS) and of the KS-related human herpesvirus (HHV-8) among homosexual men who seroconverted for HIV between 1984 and 1997. METHODS: The study participants were 387 homosexual men. Changes over a period of time were assessed by estimating KS incidence rates per 1000 person-years for the periods 1984-1989, 1990-1992, 1993-1995, and 1996-1997. The proportional incidence of KS as the AIDS-defining disease for the same periods was also calculated. To evaluate a cohort effect of calendar period, Kaplan-Meier curves were used to estimate the risk of KS by period of HIV seroconversion [i.e. before 1990 (median year of seroconversion) versus later]. Relative hazards for the four periods were estimated using competitive-risks models. We also estimated HHV-8 seroprevalence over the study period. RESULTS: Forty-eight participants developed KS. Between 1984 and 1995, the incidence rate of KS per 1000 person-years increased from 3.9 to 32.8, whereas the proportional incidence decreased from 33.3 to 24.3%. The risk of developing KS after HIV seroconversion did not change when comparing the seroconversion periods (i.e. before 1990 versus later). HHV-8 seroprevalence also remained stable. The rates of KS and the relative hazards dramatically decreased after 1995. CONCLUSIONS: Although KS incidence rates increased up to 1995, the proportional incidence decreased, due to the higher increase in rates of other AIDS-defining diseases. The finding that the risk of developing KS after HIV seroconversion remained stable over time is consistent with the stable trend of HHV-8 seroprevalence. The dramatic decrease in KS incidence rates after 1995 coincides with combined antiretroviral therapy.

Does HHV-8 have a protective role on the development of HIV encephalopathy? Italian HIV-Seroconversion Study.

OBJECTIVE: To evaluate risk factors for HIV encephalopathy and whether Kaposi's sarcoma (KS) and coinfection with human herpesvirus 8 (HHV-8) protect against this disease in a cohort of HIV seroconverters. METHODS: Individuals with known dates of HIV seroconversion belonging to different HIV exposure categories (intravenous drug users, homosexual men, heterosexual contacts) were recruited by 17 clinical centers throughout Italy. Antibodies to HHV-8 lytic antigens were detected in a subgroup of participants using an immunofluorescence assay. Risk factors for HIV encephalopathy were evaluated using Cox proportional models. The association between KS or HHV-8 infection and HIV encephalopathy was evaluated using standard statistical techniques. RESULTS: During the study period, 485 of the 1,520 participants developed acquired immunodeficiency syndrome, 38 of whom developed HIV encephalopathy. HHV-8 serologic status was determined for 390 participants. Male gender, injecting drug use, and low CD4 T-cell count were associated with HIV encephalopathy; none of the 63 participants with KS developed this disease. The risk of HIV encephalopathy did not differ significantly by HHV-8 serologic status. CONCLUSIONS: HIV encephalopathy was found to be associated with male gender and intravenous drug use. The risk increased at lower CD4 T-cell counts. Although HIV encephalopathy occurred less frequently in patients with KS, no association with HHV-8 infection was found.

Biology of Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an angioproliferative disease occurring in several different clinical-epidemiological forms that, however, share the same histological traits and are all associated with infection by the human herpesvirus 8 (HHV8). KS initiates in a context of immune dysregulation characterised by CD8+ T cell activation and the production of Th1-type cytokines that induce a generalised activation of endothelial cells leading to adhesion and tissue extravasation of lympho-monocytes, spindle cell formation and angiogenesis. These phenomena are triggered or enhanced by infection with HHV8 that, in turn, is reactivated by the same cytokines. Productively-infected circulating cells are recruited into 'activated' tissue sites where HHV8 finds an optimal environment for establishing a persistent, latent infection of KS spindle cells (KSC). HHV8 dissemination is favoured by virus escape mechanisms and immune dysregulation, and leads to immune responses that are not effective against the virus but, paradoxically, exacerbates the reactive process. Although early KS is a reactive process of polyclonal nature that can regress, in time it can progress in to a true sarcoma. The progression of KS appears to be due to the deregulated expression of oncogenes and oncosuppressor genes, to the long-lasting expression of the HHV8 latency genes and, for AIDS-KS, is promoted by the proliferative and angiogenic effects of the HIV-1 Tat protein.

Episomal HPV 16 DNA isolated from a cervical carcinoma presents a partial duplication of the early region.

An invasive cervical carcinoma was found to harbor an episomal variant of human papillomavirus (HPV) type 16 DNA, with a size of about 10.1 kb. A genomic library of the tumor was constructed in bacteriophage lambda and a recombinant phage clone was isolated by screening with HPV 16 probe. Analysis by restriction mapping and Southern hybridization showed that the isolate contained a 2.2 kb duplication of the early region, which included part of E6, all E7 and part of E1 open reading frames. Possible consequences of this duplication for oncogenesis are discussed.

Expression of human herpesvirus-8 (HHV-8) encoded pathogenic genes in Kaposi's sarcoma (KS) primary lesions.

Transcription of six different HHV-8 specific mRNAs was examined in early- and late-stage KS primary lesions. Expression of the latency-associated T0.7 mRNA and of VP23 mRNA which is a specific marker of lytic/productive infection suggested that HHV-8 is secondarily recruited into the KS lesions by productively infected monocytes, macrophages. From these cells HHV-8 is transmitted to the KS spindle cells, which are latently infected. v-BCL-2, v-MCP-1 and v-IL-6 were not expressed in latently infected KS spindle cells, therefore the impact of these factors in KS pathogenesis appears to be low. By contrast, v-Cyclin D was highly expressed in almost all latently infected spindle cells and may therefore be an important factor triggering progression of late-stage KS lesions.

Prevalence, incidence and correlates of HHV-8/KSHV infection and Kaposi's sarcoma in renal and liver transplant recipients.

BACKGROUND: To determine whether the incidence of HHV-8/KSHV infection and the risk of developing KS among organ transplant recipients differ by type of organ transplanted, we calculated the rate of HHV-8/KSHV seroconversion and the risk of developing KS among renal and liver transplant recipients. METHODS: The study population consisted of renal and liver transplant recipients recruited in two transplant centres in Rome, Italy. Both pre-transplant and post-transplant serum samples were available for all participants. The prevalence of HHV-8/KSHV infection before transplantation was calculated. To determine risk factors for infection, we calculated ORs and 95% CI. Seroconversion rates (i.e. attack rates) after transplantation were also calculated. Differences in attack rates were calculated using a binomial test for proportions. RESULTS: Of the 130 participants, 21 (16.1%) were HHV-8/KSHV-positive before transplantation. Women were more likely to be infected than men, whereas no difference was observed by type of organ transplanted. Of the 109 initially negative individuals, 13 (11.9%) developed anti-HHV-8/KSHV antibodies after transplantation. The incidence of HHV-8/KSHV infection tended to be higher among liver transplant recipients. Four renal transplant recipients and none of the liver transplant recipients developed KS after transplantation. The risk of KS was higher among recipients who were already HHV-8/KSHV-positive before transplantation. CONCLUSIONS: HHV-8/KSHV seroconversion rates appear to be higher among liver transplant recipients, compared to renal transplant recipients. However, renal transplant recipients tend to have a higher risk of KS. HHV-8/KSHV reactivation appears to play a greater role on the risk of KS than incident infections.

Cell-type dependent sensitivity of herpes simplex virus 1 mutants to plaque development inhibition by an anti-gD monoclonal antibody.

Herpes simplex virus 1 (HSV-1) mutants selected in Vero cells either for resistance to plaque development inhibition (PDI) (P1, P2 and P3) or for resistance to neutralization (N1, N2, and N3) against an anti-glycoprotein D (gD) monoclonal antibody (mAb) were characterized both in Vero and BHK cells. In Vero cells P mutants were completely resistant to PDI, while N mutants showed from moderate to good resistance. In BHK cells P mutants lost their resistance to PDI, while N mutants became fully resistant. Cell type influenced the plaque size of the mutants as well. In Vero cells P mutant plaques were larger, and N mutant plaques smaller than wild type virus plaques. In BHK cells all plaques were comparable. With one exception (N2 in BHK) resistance to neutralization could be clearly appreciated at high but not at low mAb:virus particles ratio. For most of the mutants the neutralization values remained approximately the same in Vero and BHK cells. P2 and N2 mutants were more resistant to neutralization in BHK than in Vero cells. However, only for N2 mutant did the change in neutralization resistance go in the same direction as the change in PDI resistance. The results show that it is possible to dissociate the neutralizing and the PDI activities of a mAb and that the sensitivity of a virus to plaque inhibition by an anti-gD mAb is cell-type dependent.

Parallel conduction of the phase I preventive and therapeutic trials based on the Tat vaccine candidate.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug-treated individuals (http://www.hiv1tat-vaccines.info/).

Cytokine-mediated growth promotion of Kaposi's sarcoma and primary effusion lymphoma.

Kaposi's sarcoma (KS) is an angioproliferative disease particularly frequent and aggressive in patients with AIDS but occurring also in post-transplant patients or in immunocompetent individuals of certain geographic areas. At least in its early stages, KS behaves as a reactive hyperplastic process mediated by inflammatory cytokines and angiogenic factors triggered or exacerbated by human herpesvirus-8 (HHV-8) infection. The HIV Tat protein appears to be responsible for the highly aggressive nature of AIDS-KS. Over time, however, KS may evolve into a true sarcoma in association with the expression of oncogenes and/or HHV-8 latency genes endowed with growth and anti-apoptotic properties. HHV-8 infection is also associated with primary effusion lymphoma (PEL), a rare tumor that similarly develops more frequently in the setting of HIV infection. HHV-8 latency genes are likely to contribute to the neoplastic phenotype of PEL cells, whose growth in vivo may require cytokines and factors from the host, or encoded by the virus.

Cooperative DNA binding of the bovine papillomavirus E2 transcriptional activator is antagonized by truncated E2 polypeptides.

Cooperative DNA binding of the bovine papillomavirus type 1 (BPV-1) E2 transcriptional activator (E2-TA) is thought to play a role in the transcriptional synergism of multiple E2-responsive DNA elements (J. Ham, N. Dostatni, J.-M. Gauthier, and M. Yaniv, Trends Biochem. Sci. 16:440-444, 1991). Binding-equilibrium considerations show that such involvement is unlikely, thereby suggesting that the E2-TA cooperative capacity may have evolved to play other, different roles. The role of cooperative interactions in the antagonistic activity of BPV-1-positive and BPV-1-negative E2 regulatory proteins was investigated by an in vitro quantitative gel shift assay. Viral repressor E2-TR, a truncated peptide encompassing the activator DNA-binding domain, possesses a small but measurable cooperative capacity. Furthermore, the minimal E2 DNA-binding domain interacts with the activator in a positive, heterocooperative manner. As a result, the in vitro competition of full-length and truncated E2 peptides appears to be (macroscopically) noncooperative. This heterocooperative effect is probably dominant in latently infected G0-G1 cells, in which repressor E2-TR is 10- to 20-fold more abundant than the activator. The data are discussed considering the possible role of homo- and heterocooperative DNA binding in E2-conditional gene expression.