Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae.

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

Effects of mutant Vpr/Vpx on HIV-2 assembly demonstrated by immunoelectron microscopy.

The virion-associated accessory proteins Vpr and Vpx of human immunodeficiency virus type 2 (HIV-2) are required for efficient viral replication. Vpr could be important for Vpx assembly. To investigate the interaction of Vpr and Vpx with respect to the effects of reverse transcriptase (RT) activity, viral particle information and Vpx expression site directed mutagenesis was carried out to construct Vpr, Vpx and double Vpr/Vpx HIV-2 mutants. These mutants were used for infection of peripheral blood mononuclear cells (PBM), human acute lymphoblastomic leukaemia cells (CEM-CM3) and HeLa CD4+ cells. Visualization of Vpx expression was carried out using FITC and gold labelling by means of laser scanning confocal microscopy and semi quantitative immunoelectron microscopy. Intracellular and extracellular localizations of Vpx were determined by means of fine structural analysis. Up to 80-90% reduction in the RT activity, total number of viral particles, and average Vpx expression was observed after infection of target cells with the Vpr mutant strains. In addition, intracellular Vpx expression was reduced to 51.2% with the Vpr mutant. Only 0.02% Vpx expression was detected after mutation at amino acid 62. These results provide evidence that Vpr or Vpr/Vpx mutants reduce RT activity and interfere with the expression of Vpx in HIV-2 particles during viral assembly. Vpr is efficient for Vpx corporation during viral assembly.

Contrast-enhancement for the image of human immunodeficiency virus from ultrathin section by immuno electron microscopy.

A simple contrast-enhancement method is described for electron microscopic imaging of the human immunodeficiency virus (HIV) from a sample embedded in Lowicryl K4M resin, by immuneelectron microscopy. Ultrathin sections were treated with a mixture of ruthenium red dye (RR) and osmium tetroxide (OSO4). This treatment provided good contrast enhancement of the entire ultrastructural image of virus particles without the loss of immunolabelling. RR/OsO4 solution is simple to prepare and provides a better contrast than that which is achieved during conventional post-embedding immunoelectron microscopy. Treatment of ultrathin sections from low temperature-embedded samples with RR/OsO4 solution is recommended.

Vpx association with mature core structures of HIV-2.

Vpx is a virion-incorporated protein encoded by HIV-2 and certain strains of SIV which is necessary for efficient infection of lymphocytes and macrophages. To determine the location of Vpx within the HIV-2 particle, core structures were obtained from mature HIV-2 virions. Analysis of purified cores reveal the presence of monomeric Vpx as well as a nondenaturable and nonreducible higher order Vpx complex. Characterization of the core-associated Vpx complex suggests it to be a Vpx homodimer, indicative of high concentrations of Vpx. The incorporation and concentration of Vpx in HIV-2 cores support the hypothesis that it functions within the viral preintegration complex.

Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail.

Mapping of functional domains for HIV-2 gag assembly into virus-like particles.

The human immunodeficiency virus type 2 gag precursor protein, pr41, self assembles as virus-like particles (VLP) when the gag gene is expressed in insect cells. To map the functional domains for HIV-2 gag VLP formation, a series of deletion mutants was constructed by removing sequentially the C-terminal region of HIV-2 gag precursor protein and expressing the truncated gag genes in SF9 insect cells by means of recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not prevent VLP formation. However, an additional four amino acids deletion from the C-terminus, which represents 372 amino acids at the N-terminus, made gag protein fail to form VLP. There is a proline-rich region at amino acid positions 372 and 377 of HIV-2 gag. To analyze the role of these proline residues, we generated five mutants in which proline was changed sequentially into leucine. Our results showed that replacement of one or two prolines did not stop gag VLP formation, whereas replacement of all three prolines by leucine residues completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein and the zinc finger domain are dispensable for gag VLP assembly, but the presence of at least one of the three proline residues located between amino acid positions 372 and 377 of HIV-2NIH-Z is required.

An electron-lucent region within the virion distinguishes HIV-1 from HIV-2 and simian immunodeficiency virus.

Ultrastructural comparisons of immature or budding particles of human immunodeficiency virus (HIV) types 1 and 2 and simian immunodeficiency virus of macaques (SIVmac) revealed no significant difference between these genetically distinct, but related, viruses. However, a region encompassing the core of mature HIV-1 virions was found to be more electron lucent than that observed in HIV-2 and SIVmac. This ultrastructural distinction cannot be attributed to HIV-1-specific vpu, HIV-2/SIV-specific vpx, or virion-associated vpr gene products.

Highly potent and selective inhibition of human immunodeficiency virus by the bicyclam derivative JM3100.

Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.

Further evidence of icosahedral symmetry in human and simian immunodeficiency virus.

Specimens of HIV and SIV have been examined by electron microscopy, using the techniques of conventional thin sectioning, freeze-substitution, cryosectioning, and cryomicroscopy of frozen hydrated specimens. In addition freeze-drying and critical point drying were used for both shadowed replicas and scanning electron microscopy. Thin sections revealed hexagonal, pentagonal, or near-spherical profiles. Angular particles were seen in shadowed replicas and also by scanning electron microscopy. The images observed were consistent with an icosahedral shape of the virus. It is proposed that mature HIV (SIV) is an icosadeltahedron with flat triangular facets. Size measurements of the specimens showed a wide range of values for conventional embedding, but a narrow range for specimens prepared by low-temperature techniques.

Inner architecture of human and simian immunodeficiency viruses.

The cores of human and simian immunodeficiency viruses (HIV and SIV) were observed by negative staining after isolation of the core with Nonidet P40 and glutaraldehyde. Four kinds of cores were found: asymmetric and symmetric sectoral shapes, a bar shape, and a triangular shape. These results were confirmed by the examination of ultrathin sections of whole virions. In some virions, the connection between the core and the envelope was observed after freeze fracturing. Its structure was considered to be characteristic of an intermediate stage of viral maturation. The HIV-1 core was reacted with anti-HIV-1 p24 mouse monoclonal antibody.

Transdominant inhibition of wild-type human immunodeficiency virus type 2 replication by an envelope deletion mutant.

The envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) is primarily responsible for virus attachment and entry into the target cell population. We constructed an HIV-2 mutant virus containing an in-frame deletion within the putative CD4-binding sequences of the envelope glycoprotein and confirmed that the mutant envelope is unable to bind CD4 and that the mutant virus is noninfectious. To investigate whether this mutant could dominantly interfere with wild-type replication, we coexpressed proviral DNAs of both wild-type and mutant viruses in cells and assayed the production of infectious HIV-2 virions. Interference with virus replication was indeed observed with mutant DNA, and a maximal effect was achieved with 10-fold excess mutant DNA over wild-type DNA in the cotransfection experiments. The transdominant effect on virus replication does not appear to be at the level of wild-type envelope expression or gp120-CD4 interaction. Rather, the interference may be at the level of mixed-oligomer formation during progeny virus assembly and may occur by either destabilizing the multimeric structure of gp120 or forming a defective mixed multimeric gp120 which is unable to complete the receptor binding and/or postbinding events needed for infection.

Morphogenesis of recombinant HIV-2 gag core particles.

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.

[Immunodeficiency viruses]. / Os vírus da imunodeficiência.

In the first part of this article some structural and biological aspects of the HIV viruses are presented, in our opinion among the most interesting ones, connected with the AIDS viruses. Viral infection and its evolution, particularly related with infection by HIV-2, will be presented later, in the light of our experience, obtained over several years work with African people infected by the virus. The AIDS viruses are complex retroviruses, with their own identity, but also with marked structural and biological resemblances to other retroviruses, equally pathogenical for animals. The lentivirinae subfamily to which the AIDS viruses belong includes other agents, usually classified according to the host they infect. In this way, the lentiviruses of the primates contain in the same group, besides those of HIV-1 and HIV-2, viruses that infect monkeys such as SIVMAC, SIVAGM, SIVSMM, etc. The comparative study of molecular genetics and biology of human and animal retroviruses in recent years has permitted significant progress in the understanding of the possible mechanisms that lead to the Immunodepressive Acquired Syndrome that characterizes AIDS. The presence of a gene that deactivates the activated lymphocytes only present in the lentiviruses of the primates, as well as the known tropism of those viruses to CD4 lymphocytes, and not found in the other groups, are biological aspects that are pointed out. We also refer to other characteristics of HIVs such as the cytolytic and sincicial capacity of these viruses in lymphocyte culture. Finally we present an analysis of what we were able to observe in individuals infected by HIV-2 in their own and African habitat.

Human immunodeficiency virus type 2 vpx protein augments viral infectivity.

The genomes of HIV and SIV are complex and contain several accessory genes which modulate viral replication and pathogenicity. One of these genes, vpx, is unique to the HIV-2/SIV group of viruses and encodes a virion-associated protein of unknown function. To examine the function of vpx, we constructed a vpx-deficient HIV-2 proviral clone and characterized its in vitro biological properties. Following transfection into immortalized T-cell lines, vpx-mutant HIV-2 was fully replication competent and exhibited growth kinetics and cytopathic properties equivalent to wild-type HIV-2. In addition, vpx-deficient virions were indistinguishable from wild-type HIV-2 in ultrastructure, composition of major structural proteins, and reverse transcriptase activity. In PHA-stimulated normal peripheral blood mononuclear cells (PBMCs), however, vpx-deficient virus replicated at substantially lower titers and required a 100- to 1000-fold higher inoculum to establish a productive infection. This defect was localized to early events in the viral life cycle since vpx-deficient virus exhibited a 5- to 10-fold reduction in initial (single cycle) viral DNA synthesis following acute infection of primary PBMCs. Paradoxically, in long-term (9-23 months) cultures of immortalized T-cells (SupT1) continuous high level replication of vpx-deficient, but not wild-type, virus was observed, indicating less efficient viral spread and cell killing and a more attenuated phenotype of vpx-deficient HIV-2. Taken together, these results demonstrate that vpx is required for the production of fully infectious and cytopathic HIV-2 virions and that it functions early in the viral life cycle by facilitating viral entry and/or reverse transcription. The pronounced replicative defect of vpx-deficient HIV-2 in primary PBMCs but not in short-term cultures of immortalized T-cell lines emphasizes the need to characterize the properties of nonessential HIV accessory gene products in natural target cells.

Systematic classification of HIV biological subtypes on lymphocytes and monocytes/macrophages.

The growth properties and cytopathic effects of several HIV-1 and HIV-2 strains were compared between cultures on human lymphocytes and monocytes/macrophages, respectively. For some isolates (among these three paired isolates from blood and cerebrospinal fluid) replication and cytopathogenicity were comparable between lymphocytes and monocytes/macrophages (dual tropic viruses), while others showed a very specific tropism for only one cell type. Yet another subtype grew neither well on lymphocytes nor on macrophages. Taking into account the growth properties in monocytes/macrophages we propose a classification system for HIV subtypes on these cells (alpha-delta), in analogy to the nomenclature for HIV-subtyping on lymphocytes (a-d). Using this system, some prototypic viruses (LAV/HTLV-IIIB, HIV-2ROD, SIVBK28, HIV-2ALT) as well as several other HIV-1 and HIV-2 isolates were subtyped.

Human immunodeficiency virus type 2 (HIV2).

In the mid 1980's a second human retrovirus, capable of causing the acquired immunodeficiency syndrome (AIDS), was isolated from patients of West African origin. This virus, now called human immunodeficiency virus type 2 (HIV2), was found to be distinct from human immunodeficiency virus type 1 (HIV1) but closely related to simian immunodeficiency viruses (SIV). Although the genomes of HIV1 and HIV2 are similar there are significant differences in nucleotide and amino acid sequences, most marked with the envelope genes and proteins. Both viruses, however, bind to the same CD4 cellular receptor. HIV2 is largely confined to West Africa where it is the dominant HIV, though patients infected with HIV2 have been described in Europe and America. Its transmission, clinical features and immunological effects are similar to those associated with HIV1 infection. However, there is some suggestion that the incubation period from infection to clinical disease may be longer than with HIV1 and that HIV2 may be less pathogenic. Patients with sera that react with both HIV1 and HIV2 antigens have been described, but it is unclear whether this represents serological cross reactivity or true double virus infection. Testing for HIV2 antibodies may become increasingly necessary in HIV2 non-endemic areas.