Control of viral replication and disease onset in cynomolgus monkeys by HIV-1 TAT vaccine

The Tat protein of HIV is produced early after infection and it is essential for viral replication and transmission. Tat is released by infected lymphocytes and is detected in the serum of HIV-infected patients. Extracellular Tat enters cells, where promotes HIV replication. Several studies suggest that humoral and cellular anti-Tat immunity have a protective role and may control disease progression. Of importance, Tat is conserved in its immunogenic regions among all viral subtypes except O subtype. Thus, the immunization with Tat cannot block virus entry but might block HIV replication and progression to disease. To test this hypothesis, monkeys (Macaca fascicularis) were immunized with a biologically active Tat protein. Tat was non toxic and induced specific humoral and cellular immune responses. High titers of anti-Tat antibodies capable of neutralizing Tat activity and the in vitro infection with the SHIV89.6P, Tat-specific proliferation, CTLs, TNFalpha production and skin tests were detected in the vaccinated monkeys. Most importantly, upon challenge with the highly pathogenic SHIV89.6P (10 MID50, i.v.), 5/7 of the vaccinated monkeys showed no signs of infection nor CD4+-T cell decline over a 19 months of follow-up, whereas 3/3 controls were highly infected. Thus, a Tat-vaccine is capable of controlling the acute phase of infection in nonhuman primates. These data open new avenues for the development of an AIDS vaccine.     

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.     

Pentosan polysulfate as an inhibitor of extracellular HIV-1 Tat

HIV-1 Tat protein, released from HIV-infected cells, may act as a pleiotropic heparin-binding growth factor. From this observation, extracellular Tat has been implicated in the pathogenesis of AIDS and of AIDS-associated pathologies. Here we demonstrate that the heparin analog pentosan polysulfate (PPS) inhibits the interaction of glutathione S-transferase (GST)-Tat protein with heparin immobilized to a BIAcore sensor chip. Competition experiments showed that Tat-PPS interaction occurs with high affinity (K(d) = 9.0 nm). Also, GST.Tat prevents the binding of [(3)H]heparin to GST.Tat immobilized to glutathione-agarose beads. In vitro, PPS inhibits GST.Tat internalization and, consequently, HIV-1 long terminal repeat transactivation in HL3T1 cells. Also, PPS inhibits cell surface interaction and mitogenic activity of GST.Tat in murine adenocarcinoma T53 Tat-less cells. In all assays, PPS exerts its Tat antagonist activity with an ID(50) equal to approximately 1.0 nm. In vivo, PPS inhibits the neovascularization induced by GST.Tat or by Tat-overexpressing T53 cells in the chick embryo chorioallantoic membrane. In conclusion, PPS binds Tat protein and inhibits its cell surface interaction, internalization, and biological activity in vitro and in vivo. PPS may represent a prototypic molecule for the development of novel Tat antagonists with therapeutic implications in AIDS and AIDS-associated pathologies, including Kaposi's sarcoma.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还     

A review of HIV-1 Tat protein biological effects

The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations.     

Furin cleavage of the HIV-1 Tat protein

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.     

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

Unperturbed Posttranscriptional Regulatory Rev Protein Function and HIV-1 Replication in Astrocytes

Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase) and TRBP (anti-PKR) in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.