Didier Trono - US grants

The Vif protein is essential to HIV-1 infectivity. Viruses defective in vif are poorly infectious as cell-free virions. The importance of cell- free virus was illustrated early on by the spread of AIDS in the hemophiliac population, and is still probably a key to the high rate of contamination from infected mother to fetus. Moreover, recent studies have revealed unexpectedly high levels of plasma viremia at various stages of HIV-1-induced disease, further emphasizing the role of cell-free virus in propagating the AIDS epidemic. The vif gene is found in all primate lentiviruses, as well as in other lentiviruses like FIV, BIV and visna virus, but not in classical retroviruses. This raises the possibility that Vif participates in determining the particular behavior of lentiviruses in the host, that is, their ability to establish slowly progressive infections, despite host defences, and to induce chronic degenerative diseases. This application proposes to define the role of HIV-1 Vif in determining virus infectivity. Preliminary studies presented here suggest that the function of Vif is to permit gp160-mediated viral entry. A priority will be to confirm this result, which has important implications for orienting future experiments. To analyze the role of Vif in HIV-1 replication, a combination of analytical and functional studies will be undertaken. Biochemicals properties of the Vif protein will be determined. Specifically, the intracellular localization of Vif, its potential presence in virions, as well as its possible actions on the viral envelope glycoproteins or other factors will be defined. Functional tests will search for viral or cellular factors that alter the need for Vif. Others will look at the interchangeability between Vif proteins from various viruses, to define whether Vif acts through a cellular or a virus-specific target. In addition, the mechanisms of action of Vif will be examined by comparing wtVif and deltaVif viruses through the early steps of the infectious cycle: CD4 binding, gp120/gp41 dissociation, virus internalization, uncoating, reverse transcription. Finally, potential effects of Vif on HIV-infected cells will be investigated, in the context of vif-expressing cell lines. Once the role of Vif will have been defined, rapid assays will be used to delineate Vif functional domains. Agonists of Vif will also be searched, based on analogies with proteins known to accomplish similar functions. We strongly feel that the proposed experiments will reveal crucial information regarding the way HIV is transmitted, and induces disease in patients. Most importantly, these studies might uncover new ways of blocking HIV-1 infectivity, and therefore of controlling the spread of AIDS.

This application describes studies aimed at elucidating the role of the HIV-1 Nef protein. The conservation of this gene product in primate lentiviruses, as well as experiments done in the SIV system, indicate that Nef is essential for the persistence of these viruses in the infected host, and for the pathogenesis of AIDS. Yet, the mechanisms of action of Nef remain a mystery, hampering the rational development of therapies which could target this viral protein. In the past, a number of studies have evaluated the effects of Nef on the in vitro growth of HIV. These experiments have generated a plethora of contradictory results, triggering an often bitter scientific controversy. Novel approaches are obviously needed to understand the function of Nef. This application proposes to concentrate on the effects exerted by Nef on the T helper cell, the main target of HIV. Our and other people's work show that Nef dramatically downregulates the cell surface expression of CD4, which is the main HIV receptor, but also plays a major role in immune recognition. The study of this phenomenon will be used as a guide to explore the roles of Nef, its potential ability to interfere with T cell function, and its influences on the life-cycle of HIV. The planned experiments will address the following questions: 1. What are the mechanisms of Nef-induced CD4 downregulation? 2. Does Nef influence the response of T cells to antigens or other stimuli? How? 3. What are the functional domains of Nef? 4. How does Nef-induced CD4 downregulation influence reinfection and envelope-CD4 interactions?

The Vif protein is essential to HIV-1 infectivity. Viruses defective in vif are poorly infectious as cell-free virions. The importance of cell- free virus was illustrated early on by the spread of AIDS in the hemophiliac population, and is still probably a key to the high rate of contamination from infected mother to fetus. Moreover, recent studies have revealed unexpectedly high levels of plasma viremia at various stages of HIV-1-induced disease, further emphasizing the role of cell-free virus in propagating the AIDS epidemic. The vif gene is found in all primate lentiviruses, as well as in other lentiviruses like FIV, BIV and visna virus, but not in classical retroviruses. This raises the possibility that Vif participates in determining the particular behavior of lentiviruses in the host, that is, their ability to establish slowly progressive infections, despite host defences, and to induce chronic degenerative diseases. This application proposes to define the role of HIV-1 Vif in determining virus infectivity. Preliminary studies presented here suggest that the function of Vif is to permit gp160-mediated viral entry. A priority will be to confirm this result, which has important implications for orienting future experiments. To analyze the role of Vif in HIV-1 replication, a combination of analytical and functional studies will be undertaken. Biochemicals properties of the Vif protein will be determined. Specifically, the intracellular localization of Vif, its potential presence in virions, as well as its possible actions on the viral envelope glycoproteins or other factors will be defined. Functional tests will search for viral or cellular factors that alter the need for Vif. Others will look at the interchangeability between Vif proteins from various viruses, to define whether Vif acts through a cellular or a virus-specific target. In addition, the mechanisms of action of Vif will be examined by comparing wtVif and deltaVif viruses through the early steps of the infectious cycle: CD4 binding, gp120/gp41 dissociation, virus internalization, uncoating, reverse transcription. Finally, potential effects of Vif on HIV-infected cells will be investigated, in the context of vif-expressing cell lines. Once the role of Vif will have been defined, rapid assays will be used to delineate Vif functional domains. Agonists of Vif will also be searched, based on analogies with proteins known to accomplish similar functions. We strongly feel that the proposed experiments will reveal crucial information regarding the way HIV is transmitted, and induces disease in patients. Most importantly, these studies might uncover new ways of blocking HIV-1 infectivity, and therefore of controlling the spread of AIDS.

lentiviruses, including the human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect non-dividing cells. Because at any given time most T lymphocytes in the body are quiescent, and because tissue macrophages constitute the other important virus reservoir in a patient, it is likely that this property is essential for the spread of the virus in HIV-1 infected individuals, and for AIDS pathogenesis. As such, it constitutes a suitable target for novel antiviral therapies, whose development will require understanding the molecular events involved in this process. The proposed studies aim at providing this knowledge, by dissecting the mechanisms by which HIV-1 infects non-proliferating cells. Our preliminary work has shown that critical determinants in the viral MA and Vpr proteins are essential for HIV-1 infection of macrophages and growth-arrested cells, as well as for the establishment of a stable infection intermediate in quiescent T lymphocytes. We will extend these findings, by concentrating on the following specific aims; 1) Characterize the infection intermediate established by HIV-1 in quiescent T lymphocytes, as it is likely to play a pivotal role in the spread of the virus in vivo. 2) Map all the elements which participate in allowing HIV-1 infection of non-dividing cells. 3) Determine the significance of MA phosphorylation, a modification which we show is a key regulator of HIV-1 nuclear import. 4) Further investigate the contribution of Vpr to HIV-1 infection of non- proliferating cells, and elucidate Vpr mechanism of action. 5) Pursue studies which have allowed to detect a cellular MA-binding protein, possibly a chaperone responsible for escorting HIV-1 to the nucleus of non-dividing cells.

This application describes studies aimed at elucidating the mechanism by which the HIV-1 Nef protein induces CD4 endocytosis. The conservation of nef in primate lentiviruses, as well as experiments done in the SIV system, indicate the it is essential for the persistence of these viruses in the infected host, and for the pathogenesis of AIDS. Yet, the mechanisms of Nef action remain a mystery, hampering the rational development of therapies which could target this viral protein. Work performed in the laboratory of Dr. Trono, the U.S. Principal Investigator, has demonstrated that nef triggers the rapid internalization of CD4, which is the main HIV receptor, but also plays a major role in immune recognition. This application proposes to further dissect this phenomenon, through an intensified collaboration between Dr. Trono's group and that headed by Dr. Jean-Louis Carpentier, from the Department of Morphology at the University of Geneva, Switzerland. Dr. Carpentier's expertise int he field of endocytosis will allow: 1. To perform an ultrastructural analysis of CD4 endocytosis, in the presence or the absence of Nef. 2. To analyze Nef- mediated CD4 internalization in the context of a cell-free assay for endocytosis, recently developed in his laboratory. It is strongly hoped that these studies will lead to major breakthroughs in the understanding of Nef function, as well as of CD4 metabolism. Ultimately, new therapeutic approaches for HIV might be suggested by these experiments.