FORPROTECT

BAVARIAN RESEARCH ASSOCIATION FOR INFECTION PROTECTION BY MEANS OF NEW DIAGNOSTIC AND THERAPEUTIC METHODS

DT-8 HIV-1 Vpr as a target for antiviral therapy

Since the beginning of the 1980s millions of people have been infected with human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), a deadly and so far incurable disease. As of July 2004, approximately two decades after the first clinical evidence of AIDS was reported, the United Nations estimate that more than 20 million people have died from AIDS, with 37.8 million HIV-1 infected individuals living. Although HIV-1 remains a major devastating disease, there is little hope that an effective vaccine will be developed anytime soon, nor are there efficient mechanisms available to stimulate the natural immunity against HIV-1. Another major drawback is the fact that the virus continues to persist even during prolonged therapy in latently infected host cells, classified as the viral reservoirs in vivo. Current anti-retroviral treatment is based on drugs that target either the viral enzymes, protease (PR) and reverse transcriptase (RT), or the envelope (Env) protein mediated entry of the virus. Although introduction of highly active antiretroviral therapy (HAART) in the mid nineties has led to a significant reduction in morbidity and mortality, an eradication of the virus from HIV-1 infected individuals has never been achieved. In addition, these antiviral drugs can induce severe adverse effects, particularly when administered in combination and over prolonged treatment periods. A drawback to these treatments is that with HIV-1’s high rate of mutation and replication dynamic, drug-resistant mutants are evolving. Cellular genes have much lower mutation rates, and a potential solution to this problem is to target cellular factors, enzymes or complex mechanisms that are essential for replication of HIV-1 in the host cells. More recently, we have initiated structural and functional analyses of the HIV-1 accessory protein, the viral protein R (Vpr) of HIV-1. The 96 amino acid Vpr has multiple functions in HIV-1 pathogenesis, including virion incorporation, nuclear translocation of the HIV-1 preintegration complex, nucleocytoplasmic shuttling, transcriptional activation of both the HIV-1 long terminal repeat and host genes, and induction of cell cycle arrest at the G2/M phase. A unique protocol for high-yield production of synthetic Vpr was established that has permitted various structural and functional studies. Folding and self-association of Vpr was analyzed using circular dichroism, dynamic light scattering, infrared and 1H NMR spectroscopy. Similar to native Vpr, the peptide incorporates into virus particles, activates HIV-1 replication, transduces various cell types, localizes to the nucleus and induces G2 arrest. These findings raise the possibility that circulating soluble forms of Vpr detected in peripheral blood of HIV-1 infected patients may exert biological effects on a broad range of host cells. Novel strategies to neutralize cell free Vpr will be elaborated by studying structural constraints that govern specific interaction of Vpr with viral and cellular factors. In addition, we have demonstrated that cellular uptake of Vpr occurs in a variety of cell types, even those that are not infected by HIV-1, and that this process occurs independent of cellular receptors and the viral context. As a result of our finding that Vpr represents another example of a growing list of so called “Trojan peptides”, we now follow the hypothesis that Vpr, which was detected in the blood and spinal liquor of AIDS patients, might contribute to AIDS associated diseases like the HIV lipodystrophy syndrome (HLS). HLS is a highly prevalent, complex, unexplained and currently untreatable metabolic disorder that has a severe clinical and economic impact on HIV medicine. HIV-1 Vpr is likely to be one etiologic factor in the development of HIV lipodystrophy, by virtue of its abilities to circulate in the extracellular space after release from cells containing slow-replicating HIV-1, to transduce a variety of cell types and to co-activate the glucocorticoid receptor. Vpr is the major virion-associated accessory protein that is specifically encapsidated in budding virions due to its interaction with HIV-1 Gag. Our recent investigation of the conformational heterogeneity of the proline residues in the N-terminus of Vpr by NMR experiments suggested a functional interaction between Vpr and a host peptidyl-prolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Subsequently, we identified an interaction between Vpr and the major PPIase cyclophilin A (CypA) that is also encapsidated into virions. We observed that CypA regulates the de novo synthesis of Vpr and that this hitherto unappreciated function of CypA involves prolines 5,10,14, and 35 comprising a CypA binding motif in Vpr. To unravel the underlying mechanism of this phenomenon we studied alanine-scanning mutants in the context of Vpr: prolines which form the predicted CypA binding motif are necessary and required for the responsiveness of Vpr expression to CypA inhibition. Mutational analyses in CCR5-tropic and CXCR4-tropic HIV-1 viruses now reveal a critical role of CypA for replication in macrophages, but not for T-cells. The cumulative results show that in addition to the established interaction between CypA and HIV-1 capsid, which is important during virus entry, we found that CypA is also important for de novo synthesis and thus, for the steady state level of Vpr in cell and virus. This explains why in the absence of this interaction, HIV-1 replication is reduced in macrophages where Vpr is known to be essential for productive infection. Since Vpr is present in several intra- and extracellular compartments we will further identify Vpr interacting host factors which might lead to a target specific design of Vpr inhibitors.