Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

Identification of simian immunodeficiency virus SIVMAC env gene products.

A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.     

The vpx gene of simian immunodeficiency virus facilitates efficient viral replication in fresh lymphocytes and macrophage.

vpx is a unique open reading frame found in simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) but not in HIV-1. It encodes a 12- to 16-kDa virion-associated protein. Although vpx is dispensable for viral replication in several established human lymphocyte cell lines, there is no consensus regarding whether this gene is required for efficient viral replication in freshly isolated lymphocytes. We report here that the vpx mutant of SIVmac exhibits different degrees of impairment from wild-type SIVmac in freshly isolated lymphocytes. This defect is more pronounced in macrophages from the same donors. Our findings suggest that vpx is required for efficient viral replication in fresh lymphocytes and macrophages.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus env proteins possess a functionally conserved assembly domain.

The envelope (env) glycoproteins of human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) form dimers shortly after synthesis. Analysis of the simian immunodeficiency virus (SIV) env protein expressed by a recombinant vaccinia virus revealed that it, too, forms stable homodimers. When the HIV-1 and SIV env proteins or the HIV-1 and HIV-2 env proteins were coexpressed in the same cells, heterodimers were formed. Thus, the env proteins of HIV-1, HIV-2, and SIV possess a functionally conserved domain involved in subunit-subunit recognition and assembly that likely involves the ectodomain of gp41.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

Characterization of a novel vpu-harboring simian immunodeficiency virus from a Dent's Mona monkey (Cercopithecus mona denti)

We report the identification of a new simian immunodeficiency virus (SIV), designated SIVden, in a naturally infected Dent's Mona monkey (Cercopithecus mona denti), which was kept as pet in Kinshasa, capital of the Democratic Republic of Congo. SIVden is genetically distinct from the previously characterized primate lentiviruses. Analysis of the full-length genomic sequence revealed the presence of a vpu open reading frame. This gene is also found in the virus lineage of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus (SIVcpz) and was recently described in viruses isolated from Cercopithecus nictitans, Cercopithecus mona, and Cercopithecus cephus. The SIVden vpu coding region is shorter than the HIV-1/SIVcpz and the SIVgsn, SIVmon, and SIVmus counterparts. Unlike Pan troglodytes schweinfurthii viruses (SIVcpzPts) and Cercopithecus monkey viruses (SIVgsn, SIVmon, and SIVmus), the SIVden Vpu contains the characteristic DSGXES motif which was shown to be involved in Vpu-mediated CD4 and IkappaBalpha proteolysis in HIV-1 infected cells. Although it harbors a vpu gene, SIVden is phylogenetically closer to SIVdeb isolated from De Brazza's monkeys (Cercopithecus neglectus), which lacks a vpu gene, than to Cercopithecus monkey viruses, which harbor a vpu sequence.     

Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection.

Studies with the simian immunodeficiency virus have shown that nef deletion results in a low level of viremia and a lack of disease progression in monkeys. Given the similarity of this clinical profile to that observed in long-term survivors of human immunodeficiency virus type 1 (HIV-1) infection, we sought to examine the nef gene in 10 patients who are clinically healthy and immunologically normal despite 12 to 15 years of infection. PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells obtained from long-term survivors. We found that there is no gross deletion within nef in the cases studied; most nef sequences (91.1%) obtained from 10 subjects contained a full-length and intact open reading frame. In addition, at the protein level, there were no discernible differences between the Nef consensus sequences derived from long-term survivors and those from patients with AIDS. We therefore conclude that deletion of or gross sequence abnormality within nef is not likely to be a common explanation for the well-being of long-term survivors of HIV-1 infection. Moreover, phylogenetic analysis of nef sequences suggests that HIV-1 strains found in our study subjects do not have a common origin.     

Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu, env, and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison, Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences, Nef inhibited superinfection more efficiently than Vpu and Env. Notably, nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus, maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally, we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly, one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production.     

Human immunodeficiency virus type 1 nef quasispecies in pathological tissue.

The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-1-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nef quasispecies in pathologic tissues during HIV-1 infection of the human host.     

Chimeric human immunodeficiency virus type 1 virions that contain the simian immunodeficiency virus nef gene are cyclosporin A resistant

We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.     

Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1.

Antisense RNA, transcribed intracellularly from constitutive expression cassettes, inhibits the replication of the human immunodeficiency virus type 1 (HIV-1) as demonstrated by a quantitative microinjection assay in human SW480 cells. Infectious proviral HIV-1 DNA was co-microinjected together with a fivefold molar excess of plasmids expressing antisense RNA complementary to a set of ten different HIV-1 target regions. The most inhibitory antisense RNA expression plasmids were targeted against a 1 kb region within the gag open reading frame and against a 562 base region containing the coding sequences for the regulatory viral proteins tat and rev. Experimental evidence is presented that the antisense principle is the inhibitory mechanism in this assay system.     

Expression of human immunodeficiency virus genes using baculovirus expression system

The structural protein genes of HIV-1 and HIV-2 have been expressed inSpodoptera frugiperda (SF) cells using baculovirus expression system. The noncoding flanking sequences of HIV structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. The coding sequences of thegag, pol, env, andvif proteins were inserted intoAutographa californica nuclear polyhedrosis virus (AcNPV) so that HIV genes were under the control of the AcNPV polyhedrin promoter. All recombinant AcNPV-infected SF cells express high levels of HIV structural proteins. Detailed strategies of recombinant AcNPV construction for high level protein expression are presented.     

Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates.

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.     

Chimeric human immunodeficiency virus type 1 (HIV-1) virions containing HIV-2 or simian immunodeficiency virus Nef are resistant to cyclosporine treatment

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function.     

Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus.

Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame.