Human Immunodeficiency Virus Type 1 Gag Research Articles

A simple procedure to generate chimeric Pr55gag virus-like particles expressing the principal neutralization domain of human immunodeficiency virus type

The Pr55gag human immunodeficiency virus type 1 (HIV-1) precursor protein that is capable of auto-assembling was used as a carrier for a consensus sequence of the principal neutralization domain (PND) of the HIV-1 envelope. For this purpose, a modified HIV-1 gag gene with deletion of the sequence encoding a previously described p24 epitope (amino acids 196-228 of Pr55gag) was first obtained using PCR with degenerate primers, and then cloned. This deleted gag gene allowed in a second time the insertion of a synthetic oligonucleotide cassette encoding the North American/European consensus PND precisely in place of the p24 epitope. The chimeric gene was then inserted into a baculovirus transfer vector and expressed in insect cells. The construct formed 100-140 nm virus-like particles that were released into the extracellular medium. The use of a serum-free medium that supports growth of insect cells facilitated the downstream purification of the extracellular particles. The chimeric particles were recognized by monoclonal antibodies directed to V3 by Western blot but not by immune electron microscopy, suggesting that, although the inserted sequence was still antigenic it was not exposed at the surface of the particles. The results show the ability of Pr55gag to serve as a carrier for easy insertion, in a precisely defined region, of selected epitopes of gp120 surface envelope protein, and to still auto-assemble in virus-like particles. However, the data indicate that exposed epitopes of the mature p24 protein are not presented similarly in the Pr55 precursor, and therefore that different constructs with various insertions in different places must be generated. Such constructs offer an attractive approach for HIV vaccine development and will need evaluation for both antigenicity and immunogenicity.

Antiviral properties of aminodiol inhibitors against human immunodeficiency virus and protease

A series of aminodiol inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were identified by using an in vitro peptide cleavage assay. BMS 182,193, BMS 186,318, and BMS 187,071 protected cells against HIV-1, HIV-2, and simian immunodeficiency virus infections, with 50% effective doses ranging from 0.05 to 0.33 microM, while having no inhibitory effect on cells infected with unrelated viruses. These compounds were also effective in inhibiting p24 production in peripheral blood mononuclear cells infected with HIV-1 IIIB and against the zidovudine-resistant HIV-1 strain A018C. Time-of-addition studies indicated that BMS 182,193 could be added as late as 27 h after infection and still retain its antiviral activity. To directly show that the activity of these compounds in culture was due to inhibition of proteolytic cleavage, the levels of HIV-1 gag processing in chronically infected cells were monitored by Western blot (immunoblot) analysis. All compounds blocked the processing of p55 in a dose-dependent manner, with 50% effective doses of 0.4 to 2.4 microM. To examine the reversibility of BMS 186,318, chronically infected CEM-SS cells were treated with drug and virions purified from the culture medium. Incubation of HIV-1 particles in drug-free medium indicated that inhibition of p55 proteolysis was slowly reversible. The potent inhibition of HIV-1 during both acute and chronic infections indicates that these aminodiol compounds are effective anti-HIV-1 compounds.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

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.

High frequency of Gag- and envelope-specific cytotoxic T lymphocyte precursors in children with vertically acquired human immunodeficiency virus type 1 infection.

Circulating human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) are seen less frequently in unstimulated peripheral blood mononuclear cells (PBMC) from children with vertically acquired HIV infection than in PBMC from HIV-infected adults. HIV-1 Gag-, reverse transcriptase (RT)-, and envelope (Env)-specific cytotoxic activity was studied in PBMC from HIV-infected children. Only 9% of subjects had Gag- or RT-specific CTL in unstimulated PBMC. However, in PBMC studied after CD3 stimulation, Gag- and Env-specific CTL were found in PBMC from 91% and 78% of HIV-infected children, respectively. Limiting dilution analysis of precursor CTL (pCTL) frequencies in PBMC from children > 12 months old demonstrated Gag- and Env-specific pCTL frequencies from 0.5 to 6.3/10,000 PBMC and from 0.66 to 33.0/10,000 PBMC, respectively. Thus, children with vertically acquired HIV infection have high frequencies of HIV-specific pCTL.

Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.

Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.

Virologic and immunologic studies were performed on five patients presenting with primary human immunodeficiency virus type 1 (HIV-1) infection. CD8+ cytotoxic T lymphocyte (CTL) precursors specific for cells expressing antigens of HIV-1 Gag, Pol, and Env were detected at or within 3 weeks of presentation in four of the five patients and were detected in all five patients by 3 to 6 months after presentation. The one patient with an absent initial CTL response had prolonged symptoms, persistent viremia, and low CD4+ T-cell count. Neutralizing antibody activity was absent at the time of presentation in all five patients. These findings suggest that cellular immunity is involved in the initial control of virus replication in primary HIV-1 infection and indicate a role for CTL in protective immunity to HIV-1 in vivo.

Characterization of human immunodeficiency virus type 1 Pr55gag membrane association in a cell-free system: requirement for a C-terminal domain.

Association of the human immunodeficiency virus type 1 (HIV-1) gag polyprotein precursor with cellular membranes is necessary for assembly of virions. We used in vitro synthesized HIV-1 gag to study its association with isolated cellular membranes. Rabbit reticulocyte lysates programmed with HIV-1 gag mRNA incorporated [35S]methionine and [3H]myristate into two predominant species of 55 kDa and 40 kDa. Radioimmunoprecipitation with HIV-1-specific antibodies suggested that the 55-kDa protein represented the polyprotein precursor (Pr55gag), while the 40-kDa protein was a mixture of N- or C-terminal truncations of the gag precursor. The Pr55gag protein bound to cellular membranes, while the 40-kDa mixed protein species did not. Membrane binding studies with C terminus-truncated and point mutants revealed that the seven-amino acid sequence located between the two Cys-His arrays in the nucleocapsid region was necessary for stable association to occur. Therefore, we propose that signals in addition to myristate are required for the membrane association of HIV-1 gag proteins and that these signals include a domain in the nucleocapsid protein.

Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.

A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection.

One strategy for somatic gene therapy for human immunodeficiency virus type 1 (HIV-1) infection is based on the regulated expression of dominant negative mutants of the HIV-1 gag gene. To limit expression of the mutant Gag polypeptide to HIV-1-infected cells, we have constructed a replication-defective retroviral vector that contains a Rev-responsive element. By using this construct we have obviated problems that can be associated with constitutive expression of an exogenous gene, an important step toward developing a human therapy. In uncloned T lymphocytes infected (transduced) with this retroviral construct, HIV-1 replication was inhibited by 94% with a concomitant decrease in the cytopathic effects of the virus. In addition, simian immunodeficiency virus (SIV) replication was also shown to be significantly inhibited, suggesting that this mutant Gag protein may have antiviral efficacy against a broad range of primate lentiviruses and that an SIV/macaque model can be used for further in vivo studies. These results have important implications in assessing the potential of somatic gene therapy in the treatment of HIV-1 infection.

Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases

Mutations, designed by analysis of the crystal structures of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) protease (PR), were introduced into the substrate binding pocket of RSV PR. The mutations substituted nonconserved residues of RSV PR, located within 10 A of the substrate, for those in structurally equivalent positions of HIV-1 PR. Changes in the activity of purified mutants were detected in vitro by following cleavage of synthetic peptides representing wild-type and modified RSV and HIV-1 gag and pol polyprotein cleavage sites. Substituting threonine for valine 104 (V104T), S107N, I44V, Q63M or deletion of residues 61-63 produced enzymes that were 2.5-7-fold more active than the wild type RSV PR. Substituting I42D, M73V, and A100L produced enzymes with lower activity, whereas a mutant that included both M73V and A100L was as active as wild type. Several substitutions altered the specificity for substrate. These include I42D and I44V, which contribute to the S2 and S2' subsites. These proteins exhibited HIV-1 PR specificity for P2- or P2'-modified peptide substrates but unchanged specificity with P4-, P3-, P1-, P1'-, and P3'-modified substrates. Changes in specificity in the S4 subsite were detected by deletion of residues 61-63. These results confirm the hypothesis that the subsites of the substrate binding pocket of the retroviral protease are capable of acting independently in the selection of substrate amino acids.

Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Infection with human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic viruses among leprosy patients and contacts: correlation between HIV-1 cross-reactivity and antibodies to lipoarabinomannan.

To determine the association between leprosy and human retroviral infections, 57 leprosy patients, 39 leprosy contacts, and 500 pregnant women were investigated serologically for antibodies to human immunodeficiency virus type 1 (HIV) and human T cell lymphotropic virus (HTLV) types I and II. Antibodies to Mycobacterium leprae phenolic glycolipid I (PGL-I), and lipoarabinomannan (LAM) were also analyzed. A low prevalence of HIV-1 infection was observed among leprosy patients (3.5%), leprosy contacts (0), and pregnant women (3.6%). Antibodies to HTLV-I but not -II were found more often in leprosy patients (8.7%) and contacts (12.8%) than in pregnant women (0). Sera from leprosy patients and leprosy contacts were often false-positive for HIV-1 by ELISA and were indeterminate by Western blot. LAM IgM and PGL-I IgM antibodies in sera from leprosy patients yielded significant cross-reactivities with HIV-1 pol and gag proteins. These data suggest that mycobacterial cell wall antigens may share common epitopes with HIV. Caution should be exercised when interpreting HIV-1 ELISA and Western blot data from regions where leprosy or other mycobacterial diseases are endemic.

Amplification and detection of a single molecule of human immunodeficiency virus RNA

Detection of plasma viremia in human immunodeficiency virus type 1 (HIV-1)-infected people is indispensable for the diagnosis of seronegative infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed by means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we evaluated the effects of two-step amplification with nested primers and double priming of reverse transcription on the sensitivity of RT-PCR. The sensitivity of two-step amplification was 100 times higher than that of one-step amplification. The double priming of reverse transcription further increased the sensitivity of the following two-step amplification 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2 x 10(-4) TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1 gag complementary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasma from all 22 patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and 13 out of 14 untreated asymptomatic carriers. Of 43 asymptomatic carriers under the treatment with interferon-alpha or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also noted the inhibitory effect of heparin on RT-PCR.

Enhanced Expression of Human Immunodeficiency Virus Type 1 Correlates with Development of AIDS

The progression to AIDS may be significantly related to the level of human immunodeficiency virus type 1 (HIV-1) replication. We have used quantitative cell culture and quantitative DNA and RNA PCR to measure viral load and expression in peripheral blood mononuclear cells obtained cross-sectionally and longitudinally from HIV-1-seropositive homosexual men enrolled in the Multicenter AIDS Cohort Study. Our results indicate that the number of circulating CD4 + T-lyrnphocytes producing HIV-1 increased as the total number of CD4 + T-cells declined. However, there was no correlation between the number of HIV-1-producing CD4 + cells and the duration of infection. Furthermore, the level of HIV-1 gag RNA increased as the disease progressed and CD4 + cell numbers declined. Subjects who remained asymptomatic with stable CD4 + cell counts, however, maintained a very low level of HIV-1 RNA expression during the entire period of follow-up (38-71 months). In contrast to viral RNA expression, the level of proviral DNA did not change significantly as the disease progressed. However, the level of proviral DNA was significantly higher in AIDS patients than in men who remained asymptomatic. Such increased levels of HIV-1 DNA were detected 34-68 months before the development of AIDS. These results support the role of HIV-1 RNA expression in the development of AIDS.

Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants.

We studied the effects of gag mutations on human immunodeficiency virus type 1 (HIV-1) assembly, processing, and infectivity by using a replication-defective HIV expression system. HIV mutants were screened for infectivity by transduction of a selectable marker and were examined for assembly by monitoring particle release from transfected cells. Gag protein processing and reverse transcriptase activities of mutant particles were also assayed. Surprisingly, most Gag protein mutants were assembled and processed. The two exceptions to this rule were a myristylation-minus mutant, and one gag matrix domain mutant which expressed proteins that were trapped intracellularly. Interestingly, a mutant with a 56-amino-acid deletion within the HIV gag capsid domain still could assemble and process virus particles, exhibited a wild-type retrovirus particle density, and had wild-type reverse transcriptase activity. Indeed, although most HIV-1 gag mutants were noninfectious or poorly infectious, they produced apparently normal particles which possessed significant reverse transcriptase activities. These results strongly support the notion that the HIV-1 Gag proteins are functionally involved in post-assembly, postprocessing stages of virus infectivity.

Rapid freezing of whole blood or buffy coat samples for polymerase chain reaction and cell culture analysis: application to detection of human immunodeficiency virus in blood donor and recipient repositories. The Transfusion Safety Study Group.

Storage of lymphocytes for later use in prospective epidemiologic studies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When the Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain reaction, a simple thawing and pelleting technique for recovering hemoglobin-free total white cells (WBCs) was developed. To validate the technique, parallel analysis was conducted of BCs, whole blood (WB), and PBMC samples from human immunodeficiency virus type 1 (HIV-1)-seropositive subjects. Immediate postthaw cell courts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 and 75 percent, respectively, which is as sensitive as that obtained with freshly separated PBMC lysates. Quantitative HIV-1 proviral load analysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express viral load relative to total WBCs, PBMCs, or CD4+ cells. These results establish the basis for simplified virologic analysis of cryopreserved BC or WB specimens.

Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay.

An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.

Host cell membrane proteins on human immunodeficiency virus type 1 after in vitro infection of H9 cells and blood mononuclear cells. An immuno-electron microscopic study

Human immunodeficiency virus type 1 (HIV-1)-infected H9 and blood mononuclear cells (MNCs) were studied by immunogold electron microscopy for the presence of HIV-1 gag p24 protein, env gp41 and gp120 proteins, and host cell molecules CD4, CD11a, CD25, CD54, CD63, HLA class I and HLA-DR. Uninfected H9 cells and MNC membranes labelled for CD4, HLA class I and class II, and, at low density, CD11a and CD54; lysosomal structures in the cytoplasm labelled for CD63. The infected cell surface showed immunolabelling for HIV-1 proteins, as did budding particle-like structures. Immunogold labelling of the cell membrane for CD4 was almost non-existent. The level of immunolabelling for CD11a and CD54 on infected cells was greater than that on uninfected cells; this is presumably related to a state of activation during virus synthesis. Budding particle-like structures and free virions in the intercellular space were immunogold-labelled for all host cell markers investigated. This was confirmed by double immunogold labelling using combinations of HIV-1 gag p24 labelling and labelling for the respective host cell molecule. We conclude that virions generated in HIV-1-infected cells concentrate host-derived molecules on their envelope. Also molecules with a prime function in cellular adhesion concentrate on the virion.

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.