Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site

Structure of cellular ESCRT-III spirals and their relationship to HIV budding

Abstract The ESCRT machinery along with the AAA+ ATPase Vps4 drive membrane scission for trafficking into multivesicular bodies in the endocytic pathway and for the topologically related processes of viral budding and cytokinesis, but how they accomplish this remains unclear. Using deep-etch electron microscopy, we find that endogenous ESCRT-III filaments stabilized by depleting cells of Vps4 create uniform membrane-deforming conical spirals which are assemblies of specific ESCRT-III heteropolymers. To explore functional roles for ESCRT-III filaments, we examine HIV-1 Gag-mediated budding of virus-like particles and find that depleting Vps4 traps ESCRT-III filaments around nascent Gag assemblies. Interpolating between the observed structures suggests a new role for Vps4 in separating ESCRT-III from Gag or other cargo to allow centripetal growth of a neck constricting ESCRT-III spiral. DOI: 10.7554/eLife.02184.00

Molecular Determinants That Regulate Plasma Membrane-Specific Binding of HIV-1 Structural Protein Gag.

Human Immunodeficiency Virus type 1 (HIV-1) assembly is a multistep process mediated by the viral precursor polyprotein Gag (Pr55Gag). Matrix (MA), which constitutes the N-terminal domain of Pr55Gag, is essential for membrane binding and targeting of Gag to the plasma membrane (PM). MA has a bipartite signal for membrane binding: a myristate moiety at the N-terminus and amino acid residues 17-31 that form a highly basic region (HBR) on the surface of MA. The N-terminal myristate is normally sequestered into the MA globular domain, and a structural change exposes myristate, thereby enhancing membrane binding. The HBR on the other hand is thought to bind acidic lipids. Previous results from the lab suggest that a PM-specific acidic lipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], is important for Gag localization to the PM. In this thesis, I have shown that Gag interacts specifically with PI(4,5)P2 and that this interaction is important for efficient membrane binding of Gag. To elucidate the molecular mechanisms by which Gag-PI(4,5)P2 interaction is regulated, site-directed mutagenesis was performed on the MA HBR. Using this approach, we identified three lysines that facilitate membrane binding by interacting with PI(4,5)P2. Strikingly, mutations in two other lysines in the MA HBR enhance PI(4,5)P2-independent membrane binding by exposing myristate. Thus, MA HBR has opposing roles in membrane binding. Notably, another major finding of this thesis is that RNA also negatively regulates membrane binding of Gag. In the absence but not in the presence of PI(4,5)P2, RNA bound to the MA HBR abolishes membrane binding of Gag. Overall, the results from this thesis suggest that the MA HBR regulates membrane binding both positively by binding to PI(4,5)P2 and negatively through myristate sequestration and RNA binding. This regulation ensures that Gag is targeted specifically to the PM, where it likely interacts with other viral and cellular molecules for efficient virus assembly and release.Ph.D.Microbiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86369/1/vineelac_1.pd

The Roles of Lipid and RNA in Regulating Retroviral Gag Membrane Binding and Targeting.

The HIV-1 matrix (MA) domain mediates proper Gag localization and membrane binding by interacting with phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], a plasma membrane(PM)-specific phospholipid. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine (PS), a prevalent negatively charged phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking non-specific interactions with membranes that do not contain PI(4,5)P2. In this thesis, I examined whether PI(4,5)P2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses. By comparing a panel of Gag-leucine-zipper constructs (GagLZ) containing MA of different retroviruses, I found that membrane binding mediated by retroviral MA can be broadly divided into two categories: those that are PI(4,5)P2-dependent and RNase responsive, and those that are neither. I also found that the PM-localization and virus-like particles (VLP) release of the former group is sensitive to the overexpression of a PI(4,5)P2- depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV), while the latter group is much less sensitive to 5ptaseIV overexpression. Structural analyses further suggest that the basic patch size of the retroviral MA confer susceptibility to RNA-mediated membrane binding inhibition. In my thesis, I also provided in vitro and cell-based evidence supporting that RNA-mediated suppression occurs in cells and that RNA can inhibit membrane binding of HIV-1 Gag at a concentration that is much lower than the estimated RNA concentration in the cell. Hence, RNA-mediated suppression is a physiologically relevant mechanism that prevents Gag from binding promiscuously to prevalent PS-containing membranes. Finally, I examined the roles of PI(4,5)P2 and RNA in regulating the targeting of HIV-1 Gag to the site of assembly, the virus-containing compartments (VCC), in primary macrophages. I found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression. However, HIV-1 MA contributes only to membrane binding but not in Gag targeting to the VCC. I also determined that HIV-1 nucleocapsid (NC) is important for VCC-specific localization of HIV-1 Gag. This suggests that targeting of HIV-1 Gag to the VCC adopts a different mechanism than Gag targeting to the PM in HeLa and T cells.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113324/1/arolni_1.pd

The Role of Lipids in Retrovirus Replication

Retroviruses undergo several critical steps to complete a replication cycle. These include the complex processes of virus entry, assembly, and budding that often take place at the plasma membrane of the host cell. Both virus entry and release involve membrane fusion/fission reactions between the viral envelopes and host cell membranes. Accumulating evidence indicates important roles for lipids and lipid microdomains in virus entry and egress. In this review, we outline the current understanding of the role of lipids and membrane microdomains in retroviral replication

HOST PROTEIN INCORPORATION IN HUMAN IMMUNODEFICIENCY VIRUS-1

Human Immunodeficiency Virus (HIV) incorporates a number of host proteins. These proteins can provide information on the function of viral proteins, as well as on the general process of HIV biogenesis. Determining the methods of incorporation and potential functional importance will help advance our knowledge of the HIV lifecycle and holds the potential to produce additional targets for antiretroviral therapy. Here, we used a variety of complementary techniques to determine which host proteins are incorporated into HIV particles. We found that most of the CD28 and B7 family costimulatory molecules are excluded from viral particles. Using a novel purification technique and mass spectrometry analyses, we were able to characterize host protein incorporation in HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the data sets. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, ERM proteins, EH4, a phosphodiesterase, cyclophilin A, and others. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral lifecycle. Additionally, this common protein set protein is predicted to interact with >1000 related proteins. Many of these secondary interacting proteins are reported to be incorporated into virions. Thus, only a few direct interactions between host and viral proteins may drive host protein composition in virions. We hypothesized that these may be driven by the tetraspanin family of proteins, putative membrane organizers determining the lipid and protein composition of tetraspanin enriched membranes. We found that knockdown of various tetraspanins in T cell lines did not significantly alter viral release or phenotype. Ultimately, cell type-specific differences in host protein interaction and expression may drive virion phenotypic diversity, despite conserved primary viral protein-host protein interactions across cell types. Further, the primary interactions found between viral and host proteins are likely driven by selective pressures including response to host restriction factors and membrane structural requirements

Étude du mécanisme d'incorporation sélective de l'ICAM-1 par le VIH-1 et évaluation de la sensibilité de virions porteurs d'ICAM-1 à l'action inhibitrice du T-20

Plusieurs études ont démontré que le virus d'immunodéficience humaine de type 1 (VIH-1) possède la propriété d’incorporer diverses protéines de la cellule-hôte. Certaines observations indiquent que ce processus d'incorporation est sélectif. Cependant, les mécanismes expliquant la sélectivité des protéines incorporées demeurent largement inconnus. Les travaux présentés ciblent l'identification du facteur responsable de l'incorporation sélective de la protéine cellulaire ICAM-1 par le VIH-1. Parallèlement, nous avons évalué la sensibilité des virus porteurs de l’ICAM-1 à l’action de l’inhibiteur de fusion, T-20. Un système de transfection et d'expression transitoire a été utilisé afin de produire des particules virales isogéniques avec ou sans l’ICAM-1, et de certains de ses variants. Ce système nous a permis de démontrer que le VIH-1 s’approprie la molécule d’adhésion ICAM-1 par un processus indépendant de l’enveloppe virale (Env), mais dépendant de la portion intracytoplasmique de l’ICAM-1. Cette incorporation serait dirigée par une interaction directe ou indirecte entre la molécule d’adhésion et le précurseur immature Gag du VIH-1. Par ailleurs, la présence de l’’ICAM 1 sur le VIH-1 augmente la résistance des virions à l’action du T-20 en accélérant la cinétique de fusion du virion. La présence de l’ICAM-1 à la surface des virions et son rôle dans le cycle réplicatif du VIH 1 sont très importants dans la pathogenèse du VIH-1.INTRODUCTION Previous works have indicated that incorporation of surface glycoprotein into retroviruses such as the human immunodeficiency virus type 1 (HIV-1) is not a highly specific process since several cellular glycoproteins can be inserted within the mature viral particle. The mechanism(s) that govern the acquisition of such host constituents have remained so far elusive. OBJECTIVES We have examined the molecular basis, associate to ICAM-1 localization and structural viral proteins responsible for the selective incorporation of the adhesion molecule ICAM-1 within HIV-1. We also investigated whether sensitivity to the newly developed fusion inhibitor T-20 is affected by incorporation of the adhesion molecule ICAM-1 in HIV 1. METHODS We have first investigated the role played by the viral envelope (Env) of HIV-1 in the acquisition of host ICAM-1. The incorporation process of ICAM-1 was also investigated by using different ICAM-1 constructs in which both transmembranes and intracytoplasmic tails were modified. These investigations were performed in combination with virus capture and immunoprecipitation studies, Western blot, confocal microscopy analyses, and infectivity assays. We finally used laboratory isolates of HIV-1 (X4- and R5-tropic) either lacking or bearing ICAM-1 as well as clinical variants into infectivity tests to both evaluate their respective IC50 for the fusion inhibitor and to understand the mechanism of resistance bring by the presence of this host molecule. RESULTS Mutation in the matrix (MA) domain or on Env-deficient viruses produced either in immortalized or primary human cell lines does not affect the incorporation of ICAM-1 by HIV-1. However, the incorporation seems to be conducted by the cytosolic tail of ICAM-1. Further experiments suggested that there is an association – direct or indirect – between ICAM-1 and virus-encoded Pr55Gag. We also demonstrate that ICAM-1-bearing virions are more resistant to T-20 than isogenic HIV-1 particles lacking this host adhesion molecule probably based on a reduction of the kinetic window during which the viral envelope is sensitive to T-20. CONCLUSION This study represents the first demonstration that structural Gag polyproteins mediate the uptake of a host-derived cell surface constituent (ICAM-1) by interacting directly with its cytoplasmic domain or by interacting with a partner into the cytosol. This observation describes a new strategy by which HIV-1 can modulate its replicative cycle considering that insertion of ICAM-1 within nascent virions has been shown to affect virus life cycle and also the sensitivity to the newly develop class of inhibitor including T-20