Julian W. Bess

Distribution and three-dimensional structure of AIDS virus envelope spikes

Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had ∼14 and ∼73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) ‘head’ of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein ‘stalk’ of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.

Architecture and secondary structure of an entire HIV-1 RNA genome.

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS and other serious health threats. Viral replication is regulated at many levels, including the use of conserved genomic RNA structures. Most potential regulatory elements in viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests that RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions, including splice site acceptors and hypervariable regions. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by previously unrecognized regulatory motifs and that extensive RNA structure constitutes an important component of the genetic code.

Proteomic and Biochemical Analysis of Purified Human Immunodeficiency Virus Type 1 Produced from Infected Monocyte-Derived Macrophages

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects CD4+ T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions

We used electron tomography to directly visualize trilobed presumptive envelope (env) glycoprotein structures on the surface of negatively stained HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions. Wild–type HIV-1 and SIV virions had an average of 8–10 trimers per virion, consistent with predictions based on biochemical evidence. Mutant SIVs, biochemically demonstrated to contain high levels of the viral env proteins, averaged 70–79 trimers per virion in tomograms. These correlations strongly indicate that the visualized trimers represent env spikes. The env trimers were without obvious geometric distribution pattern or preferred rotational orientation. Combined with biochemical analysis of gag/env ratios in virions, these trimer counts allow calculation of the number of gag molecules per virion, yielding an average value of ≈1,400. Virion and env dimensions were also determined. Image-averaging analysis of SIV env trimers revealed a distinct chirality and strong concordance with recent molecular models. The results directly demonstrate the presence of env trimers on the surface of AIDS virus virions, albeit at numbers much lower than generally appreciated, and have important implications for understanding virion formation, virus interactions with host cells, and virus neutralization.

Envelope Glycoprotein Incorporation, Not Shedding of Surface Envelope Glycoprotein (gp120/SU), Is the Primary Determinant of SU Content of Purified Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55°C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37°C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.

Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS

Abstract:  As new assay methods for quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), such as real time RT‐PCR techniques, approach theoretical limits of per reaction sensitivity, further increments in the sensitivity of measurements of viral load can only be achieved by increasing the amount of input RNA per reaction. We describe a robust, convenient, rapid integrated approach for specimen preparation and real time RT‐PCR assay for plasma simian immunodeficiency virus (SIV) RNA viral load that provides a threshold sensitivity of 10 copy Eq/ml, and tolerates less than optimally processed specimens. The method provides accurate quantitation of viral load for the SIV virus isolates in common use for non‐human primate studies. We demonstrate the utility of the method in sensitively tracking viral load in an animal showing effective control of viral replication to levels below the threshold for quantitation in conventional assays.

Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.

Electron Tomography of the Contact between T Cells and SIV/HIV-1: Implications for Viral Entry

The envelope glycoproteins of primate lentiviruses, including human and simian immunodeficiency viruses (HIV and SIV), are heterodimers of a transmembrane glycoprotein (usually gp41), and a surface glycoprotein (gp120), which binds CD4 on target cells to initiate viral entry. We have used electron tomography to determine the three-dimensional architectures of purified SIV virions in isolation and in contact with CD4+ target cells. The trimeric viral envelope glycoprotein surface spikes are heterogeneous in appearance and typically ∼120 Å long and ∼120 Å wide at the distal end. Docking of SIV or HIV-1 on the T cell surface occurs via a neck-shaped contact region that is ∼400 Å wide and consistently consists of a closely spaced cluster of five to seven rod-shaped features, each ∼100 Å long and ∼100 Å wide. This distinctive structure is not observed when viruses are incubated with T lymphocytes in the presence of anti-CD4 antibodies, the CCR5 antagonist TAK779, or the peptide entry inhibitor SIVmac251 C34. For virions bound to cells, few trimers were observed away from this cluster at the virion–cell interface, even in cases where virus preparations showing as many as 70 envelope glycoprotein trimers per virus particle were used. This contact zone, which we term the “entry claw”, provides a spatial context to understand the molecular mechanisms of viral entry. Determination of the molecular composition and structure of the entry claw may facilitate the identification of improved drugs for the inhibition of HIV-1 entry.

HIV-1 and microvesicles from T cells share a common glycome, arguing for a common origin

HIV-1 is a master at deceiving the immune system, usurping host biosynthetic machinery. Although HIV-1 is coated with host-derived glycoproteins only glycosylation of viral gp120 has been described. Herein we utilize lectin microarray technology to analyze the glycome of intact HIV-1 virions. We show that the glycan coat of human T-cell line-derived HIV-1 matches that of native immunomodulatory microvesicles. The carbohydrate composition of both virus and microvesicles is cell-line dependent, suggesting a mechanism to rapidly camouflage the virus within the host. In addition, binding of both virus and microvesicles to antiviral lectins is enriched over the host cell, raising concern about targeting these glycans for therapeutics. This work also sheds light on the binding of HIV-1 to galectin-1, an important human immune lectin. Overall, our work strongly supports the theory that HIV-1 co-opts the exocytic pathway of microvesicles, potentially explaining why eliciting a protective antiviral immune response is difficult.

Partial Activation and Induction of Apoptosis in CD4+ and CD8+ T Lymphocytes by Conformationally Authentic Noninfectious Human Immunodeficiency Virus Type 1

ABSTRACT Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4+ and CD8+ peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.