Kaposi sarcoma-associated herpesvirus promotes tumorigenesis by modulating the Hippo pathway.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS), an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study, we identified that YAP/TAZ, two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway, are activated in KSHV-infected cells in vitro, KS-like mouse tumors and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2, promoting the activation of YAP/TAZ. Furthermore, depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacologic inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS, and also suggests a potential of using YAP inhibitors for KS intervention.

Differential effects of HIV-1 protease inhibitors on dendritic cell immunophenotype and function.

Recent findings show that human immunodeficiency virus (HIV)-1 protease inhibitors designed to specifically inhibit the aspartic protease of HIV-1 nonetheless exert various effects on immune cell function in vitro and in vivo. Dendritic cells (DC), central players of the immune system, express several aspartic proteases that are important for DC function. In the present study, we demonstrate that all of the HIV-1 protease inhibitors tested affect DC maturation. In addition, saquinavir had a strong inhibitory effect on the T-cell stimulatory capacity of mature DC. In contrast, indinavir had only a slight effect on DC induced T-cell proliferation and allowed efficient transduction of DC with a replication-incompetent HIV-1 vector designed for DC-based immunotherapy. HIV-1 protease inhibitors that have little or no effect on DC function may be preferable for combination with immunotherapy for HIV/AIDS.

Topical treatment of cutaneous lesions of acquired immunodeficiency syndrome-related Kaposi sarcoma using alitretinoin gel: results of phase 1 and 2 trials.

OBJECTIVE: To evaluate the efficacy and safety of topical alitretinoin gel (9-cis-retinoic acid [LGD1057], Panretin gel; Ligand Pharmaceuticals, Inc, San Diego, Calif) in cutaneous Kaposi sarcoma (KS). DESIGN: Open-label, within-patient, controlled, dose-escalating phase 1 and 2 clinical trials. In all patients, 1 or more cutaneous KS lesions were treated with alitretinoin gel, and at least 2 other lesions served as untreated controls for up to 16 weeks. Alitretinoin (0.05% or 0.1% gel) was applied twice daily for the first 2 weeks and up to 4 times daily thereafter, if tolerated. SETTING: Nine academic clinical centers. PATIENTS: One hundred fifteen patients with biopsy-proven acquired immunodeficiency syndrome (AIDS)-related KS. MAIN OUTCOME MEASURES: AIDS Clinical Trials Group response criteria. RESULTS: Statistically significant clinical responses were observed in 31 (27%) of 115 patients for the group of treated index lesions compared with 13 (11%) for the group of untreated control lesions (P<.001). Responses occurred with low CD4(+) lymphocyte counts (<200 cells/microL) and in some patients with refractory response to previous systemic anti-KS therapy. The incidence of disease progression was significantly lower for treated index lesions compared with untreated control lesions (39/115 [34%] vs 53/115 [46%]; P =.02). Alitretinoin gel generally was well tolerated, with 90% of treatment-related adverse events confined to the application site and only mild or moderate in severity. CONCLUSIONS: Alitretinoin gel has significant antitumor activity as a topical treatment for AIDS-related KS lesions, substantially reduces the incidence of disease progression in treated lesions, and is generally well tolerated.

Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy.

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1 env were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1.

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.

A controlled, Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA.

This Phase I study, "Ribozyme Gene Therapy of HIV-1 Infection" (UCSD HSC #971072, FDA BB-IND 6405), is a prospective, open-label trial of infusion of autologous gene-altered cells into asymptomatic HIV-1 seropositive individuals. The objectives of this trial are to test the safety, feasibility, and potential efficacy of T-cell ribozyme gene therapy of HIV-1 infection. To accomplish this, autologous CD8-depleted mononuclear cells are transduced with ribozyme expressing or control murine retroviral vectors, expanded ex vivo, and and infused. Subjects are monitored intensively to determine effects of infusion on HIV burden and replication. In addition, in vivo survival of control and ribozyme transduced cells is followed in an effort to obtain evidence of proof of concept. A unique strategy of sample blinding is introduced in this protocol, wherein both subject and control specimens are supplied to the research laboratory as coded samples, spiking blood from HIV seropositive volunteers matched for CD4 lymphocyte count with known but varying numbers of cells transduced with each vector. While this study is still in progress, preliminary results indicate that infusion of gene-altered, activated T-cells in HIV infected patients is safe, and that transduced cells can persist for long intervals in HIV-infected subjects. Results also suggest ribozyme transduced cells may possess a survival advantage in vivo.

CXCR4 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells.

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.

Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

A minimally replicative HIV-2 live-virus vaccine protects M. nemestrina from disease after HIV-2(287) challenge.

M. nemestrina immunized with an apathogenic HIV-2 molecular clone (HIV-2KR) were protected from CD4 decline and disease upon challenge with HIV-2(287), after any immunizing virus could be detected. Higher but not lower inocula of HIV-2KR were protective against intravenous inoculation of either 10(5) or 10(1) TCID50 of HIV-2(287). Protected animals displayed substantial reductions in PBMC proviral burden (1-3 logs), viral titers (1-2 logs), and plasma viral RNA (2-4 logs) compared to unprotected or naive animals as early as 1 week postinfection. Plasma viral RNA became undetectable after 24 weeks in protected animals, but remained high in unprotected animals. No viral RNA was present in the spleen of the protected animal necropsied more than a year after challenge (though viral DNA was still present). No neutralizing responses could be demonstrated, but CTL activity was detected sooner and at higher levels after challenge in protected than in unprotected macaques. In this novel HIV-2 vaccine model, protection was clearly dose-dependent, and clearance of challenge virus RNA from the plasma did not require detectable ongoing replication of the immunizing virus at the time of challenge.

Characterization of a molecular clone of HIV type 2 infectious for Macaca nemestrina.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier.

BACKGROUND: HIV-1 invades the central nervous system early after infection when macrophage infiltration of the brain is low but myelin pallor is suggestive of blood-brain-barrier damage. High-level plasma viremia is a likely source of brain infection. To understand the invasion route, we investigated virus penetration across in vitro models with contrasting paracellular permeability subjected to TNF-alpha. MATERIALS AND METHODS: Blood-brain-barrier models constructed with human brain microvascular endothelial cells, fetal astrocytes, and collagen I or fibronectin matrix responded in a dose-related fashion to cytokines and ligands modulating paracellular permeability and cell migration. Virus penetration was measured by infectious and quantitative HIV-1 RNA assays. Barrier permeability was determined using inulin or dextran. RESULTS: Cell-free HIV-1 was retained by the blood-brain barrier with close to 100% efficiency. TNF-alpha increased virus penetration by a paracellular route in a dose-dependent manner proportionately to basal permeability. Brain endothelial cells were the main barrier to HIV-1. HIV-1 with monocytes attracted monocyte migration into the brain chamber. CONCLUSIONS: Early after the infection, the blood-brain barrier protects the brain from HIV-1. Immune mediators, such as TNF-alpha, open a paracellular route for the virus into the brain. The virus and viral proteins stimulate brain microglia and macrophages to attract monocytes into the brain. Infiltrating macrophages cause progression of HIV-1 encephalitis.

Ex vivo transduction and expansion of CD4+ lymphocytes from HIV + donors: prelude to a ribozyme gene therapy trial.

Preparations for a phase I trial of ex vivo, anti-HIV ribozyme gene therapy have included optimization of transduction and expansion of CD4+ lymphocytes from HIV-1 infected donors, using reagents suitable for production of cell products for human infusion. We also determined whether transduction by the ribozyme vector would inhibit replication and spread of endogenous HIV-1, and result in preferential survival of ribozyme-transduced CD4+ cells during lymphocyte expansion. Transduction efficiency, as estimated by DNA quantitative competitive (QC)-PCR, was similar for both control (LNL6) and ribozyme expressing (MJT) murine retroviral vectors (approximately 20%.) In the absence of antiviral agents, cells transduced with MJT exhibited three-fold greater numbers of CD4+ cells 2 weeks after transduction than did LNL6 transduced cells. In addition, viral replication was delayed 2-3 weeks in MJT transduced cultures. Both transduced cell populations expanded by 2-3 logs within 2 weeks. The clinical protocol involves infusion of both ribozyme and control vector transduced cells, making identification of agents capable of suppressing replication and spread of endogenous virus during ex vivo expansion necessary. The combination of nevirapine (100 nM) and CD4-PE40 (100 nM) completely suppressed endogenous virus replication in cultures transduced with either vector. At reduced concentrations of nevirapine, virus replication was suppressed only in MJT transduced cells.

An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.

Molecular cloning and characterization of a TAR-binding nuclear factor from T cells.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.

Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro.

To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system.

Antiproliferative effects of enediynes on AIDS-derived Kaposi's sarcoma cells.

We have investigated the antiproliferative and cytotoxic effects of selected enediynes against three Kaposi's sarcoma (KS) cell lines. The enediynes tested were found to be very potent in inhibiting the growth of KS cells. Treatment with concentrations of 10(-10) M or less were capable of producing 50% inhibition of growth. Furthermore, treatment of KS cells with enediynes induced apoptosis in up to 80% of the cells. This unique class of antitumor agents has therapeutic potential for the treatment of KS.

Antiproliferative effect of retinoid compounds on Kaposi's sarcoma cells.

A panel of retinoid compounds (tretinoin, isotretinoin, acitretin, and RO13-1470) were tested for inhibitory activity against Kaposi's sarcoma cell (KSC) cultures in vitro. Tretinoin was found to be the most effective retinoid tested, inhibiting the growth of KSC in vitro while having no effect on the expression of interleukin-6 and basic fibroblast growth factor, two important cytokines involved in KSC growth. Tretinoin also did not appear to downregulate the expression of receptors for these two cytokines. At low concentrations (10(-9) M), acitretin and tretinoin selectively inhibited growth of early passage KSC. At higher concentrations (10(-6)-10(-5) M), retinoid treatment induced a pattern of DNA degradation and morphological changes in KSC characteristic of apoptosis (programmed cell death). The inhibitory activity of tretinoin on KSC growth was decreased if human serum (but not fetal calf serum) was present in the growth medium, and partially restored by removal of serum lipids. These data suggest that retinoids possess potential as therapeutic agents in Kaposi's sarcoma.