Kinetics of processive nucleic acid polymerases and nucleases.
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DNA-Directed DNA Polymerase↗
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Blockage of N-myristoylation of HIV-1 gag induces the production of impotent progeny virus.
The role of the N-myristoylation of the human immunodeficiency virus type 1 (HIV-1) gag protein in ACH-2 cells was studied. The infectivity of HIV-1 from the cells stimulated with phorbol 12-myristate 13-acetate (PMA) was suppressed by pretreatment with N-myristoyl glycinal diethylacetal (N-Myr-GOA), a potent N-myristoylation inhibitor, and the blockage of myristoylation resulted in accumulation of immature gag precursors. The viral particles which budded from the non-N-Myr-GOA-treated ACH-2 cells stimulated with PMA exhibited a typical viral phenotype, whereas those which budded from the N-Myr-GOA-treated ACH-2 cells stimulated with PMA were twisted, as observed electron microscopically. In electron microscopic analyses with gold-labeled monoclonal antibodies to gag and env, gag and env were detected adjacent to each other in the PMA-stimulated ACH-2, but no env was detected in the cells treated with N-Myr-GOA. Taken together, the results suggest that the myristoylation of HIV-1 gag seems to be responsible for both maturation of gag and acquisition of HIV-1 infectivity.
Acetaldehyde↗
Early function of HIV-1 Gag proteins is cell-dependent.
Various gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were monitored for their replication potentials and defective replication sites in various CD4-positive T-cell lines. Some matrix, capsid, and nucleocapsid mutants displayed a replication defect in a cell-dependent manner. The single-round replication assays demonstrated that these mutants were defective at an early infection phase also in a cell-dependent way. These results indicated that interaction of a cell factor(s) and Gag proteins is involved in an early process of HIV-1 replication.
CD4-Positive T-Lymphocytes↗
A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases.
We found and isolated two natural products in the extract from a basidiomycete, Ganoderma lucidum, as eukaryotic DNA polymerase inhibitors. The compounds were identified as cerebrosides, (4E,8E)-N-D-2'-hydroxypalmitoyl- 1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and (4E,8E)-N-D-2'-hydroxystearoyl-1-O-beta-D-glucopyranos yl-9-methyl- 4,8-sphingadienine and were found to be identical to the mushroom fruiting body-inducing substances (FIS) reported. These cerebrosides selectively inhibited the activities of replicative DNA polymerases, especially the alpha-type, from phylogenetically broad eukaryotic species, whereas they hardly influenced the activities of DNA polymerase beta, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV reverse transcriptase, RNA polymerase, deoxyribonuclease I, and ATPase. The inhibition of another replicative polymerase, the delta-type, was moderate. The inhibitions of the replicative polymerases were dose-dependent, and the IC50 for animal or mushroom DNA polymerase alpha was achieved at approximately 12 micrograms/ml (16.2 microM) and for animal DNA polymerase delta at 57 micrograms/ml (77.2 microM). FIS is possibly a DNA polymerase inhibitor specific to the replicative enzyme group, and the fruiting body formation may be required for the suppression of the DNA replication or the vegetative growth of the mycelium.
Animals↗
Five viral peptide-HLA-A2 co-crystals. Simultaneous space group determination and X-ray data collection.
We prepared and crystallized five complexes of the human histocompatibility molecule HLA-A2 with peptides derived from human immunodeficiency virus type 1, human T lymphotropic virus type 1, influenza A virus and hepatitis B virus proteins. Each HLA-A2 complex was refolded in vitro from insoluble proteins produced in bacteria; to crystallize, two of the complexes required seeding with microcrystals of another complex. Maintained at -160 degrees C, single co-crystals of each of the five peptide-HLA-A2 complexes yielded complete X-ray diffraction data sets to a resolution of approximately 2.5 A. After a sufficient number of diffraction peaks were acquired during data collection, the direct analysis of integrated intensities established the point group of the co-crystal, thus allowing an efficient data collection strategy to be designed. The subsequent examination of systematic absences revealed that the five peptide-HLA-A2 co-crystals formed in space groups P1, P2(1), or P2(1)2(1)2(1). Molecular replacement structure solutions yielded unambiguous protein electron density maps, thus confirming the space group determinations. The system of obtaining HLA-A2 co-crystal structures described here is applicable to other crystallographic problems where structures of several related molecules from uncharacterized single crystals are required.
Amino Acid Sequence↗
Ranking potential binding peptides to MHC molecules by a computational threading approach.
In this paper, an approach developed to address the inverse protein folding problem is applied to prediction of potential binding peptides to a specific major histocompatibility complex (MHC) molecule. Overlapping peptides, spanning the entire protein sequence, are threaded through the backbone coordinates of a known peptide fold in the MHC groove, and their interaction energies are evaluated using statistical pairwise contact potentials. With currently available tables for pairwise potentials, promising results are obtained for MHC-peptide complexes where hydrophobic interactions predominate. By ranking the peptides in an ascending order according to their energy values, it is demonstrated that, in most cases, known antigenic peptides are highly ranked. Furthermore, predicted hierarchies are consistent with experimental binding results. Currently, predictions of potential binding peptides to a specific MHC molecule are based on the identification of allele-specific binding motifs. However, it has been demonstrated that these motifs are neither sufficient nor strictly required to ensure binding. The computational procedure presented here succeeds in determining the MHC binding potential of peptides along a protein amino acid sequence, without relying on binding motifs. The proposed scheme may significantly reduce the number of peptides to be tested, identify good binders that do not necessarily show the known allele-specific binding motifs, and identify the best candidates among those with the motifs. In general, when structural information about a protein-peptide complex is available, the current application of the threading approach can be used to screen a large library of peptides for selection of the best binders to the target protein.
Amino Acid Sequence↗
Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.
Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.
Amino Acid Sequence↗
The role of complement and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-infected patients.
The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.
Animals↗
Nucleotide sequence analysis of IS1533 from Leptospira borgpetersenii: identification and expression of two IS-encoded proteins.
The nucleotide sequence of IS1533, an insertion sequence-like element cloned from the spirochete Leptospira borgpetersenii, was determined. IS1533 contains imperfect terminal inverted repeats (IVR) of 31 bp flanking a 1402-bp internal sequence. A putative target sequence was identified, and insertion may result in duplication of 2 bp. The internal sequence has a single open reading frame (ORF). IS1533 encodes two proteins (43.5 and 41 kDa) initiating alternatively at either the first or the second AUG codons of the ORF. These proteins are related to a recently recognized family of IS-encoded transposases and bacterial recombinases, all which share a region of homology with the active site of the HIV reverse transcriptase. The IS1533-encoded proteins were expressed in Escherichia coli. Both the 43.5- and 41-kDa proteins bound IS1533 DNA probes in a Southwestern blot assay. These data suggest that one or both proteins function during transposition of IS1533.
Amino Acid Sequence↗
Inhibition of human immunodeficiency virus type 1 (HIV-1) penetration into target cells by synthetic peptides mimicking the N-terminus of the HIV-1 transmembrane glycoprotein.
To investigate the mechanism of action of the 22-amino-acid HIV fusion peptide on HIV infection, we studied its influence on virus adsorption and HIV-induced syncytium formation. The effect of the peptide preparations on the synthesis of viral antigens in HIV-infected cell cultures was determined by antigen capture assay, and the inhibition of proviral DNA synthesis was detected by hybridization with a HIV-specific oligonucleotide probe after PCR amplification. Fusion peptides inhibited HIV-induced syncytium formation and antigen production in lytic infected cells, and this effect was increased in conjugation with bovine serum albumin or with synthetic net-charged polymer by its C-terminus. The association of peptide with carrier by N-terminus, or with positive-charged polymer or gelatin completely abolished its effect on HIV infection. No peptide preparations influenced HIV-1 chronically infected cells. Because peptide preparations blocked the HIV-specific DNA synthesis 2 hr after infection without influencing virus adsorption and reverse transcription, we concluded that the block of infection occurred during the penetration of virions through the cell membrane. On the basis of results obtained we propose that our peptide preparations could be used for anti-HIV chemotherapy. The possibility of the existence of receptors for gp41 N-terminal region on target cell membrane is discussed.
Amino Acid Sequence↗
The kinetics of human immunodeficiency virus reverse transcription are slower in primary human macrophages than in a lymphoid cell line.
Reverse transcription is a critical event in the life of a retrovirus and a potential determinant of viral infectivity. The kinetics of "endogenous" reactions are relatively well defined but little is known about HIV reverse transcription during infection. In this report, we have estimated the rate and efficiency of HIV-1 reverse transcription in primary macrophages and H9 lymphoid cells using quantitative PCR to detect intermediate cDNA structures. DNA synthesis is completed 12-16 hr after infection of H9 cells, but requires more than 36 hr in M phi, owing to slower rates of extension and template switching. Reverse transcription was inefficient in both cell types and in H9 cells the kinetics were sufficiently well resolved to estimate that only one in three transcripts initiated were extended to full-length viral DNA. Slower DNA synthesis largely accounts for the longer replicative cycle of HIV in macrophages. The rate of reverse transcription, which may depend upon levels of deoxynucleotide triphosphate substrates, is a potential factor in modulating the permissiveness of macrophage populations in vivo.
Base Sequence↗
Restriction of HIV-1 replication in intestinal cells is genetically controlled by the gag-pol region of the HIV-1 genome.
The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis, HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment BssHII255-EcoRI4183) genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level.
Cell Line↗
HIV infection of choriocarcinoma cell lines derived from human placenta: the role of membrane CD4 and Fc-Rs into HIV entry.
We have shown previously that trophoblast cells from human placenta can be infected with HIV-1 and a productive infection established. Recently, (1991, J. Virol. 65, 2102-2107) Zachar et al. and D. M. Phillips and X. Tan (1992, AIDS Res. Hum. Retroviruses 8, 1697-1705) have described in vitro infection of choriocarcinoma cell lines. Using choriocarcinoma cell lines (JAR, BeWo, and FD25, a trophoblast-derived cell line) we have infected these cells with several laboratory strains of virus and have shown that this can be prevented either by sCD4 or by antibodies to CD4. This provides strong evidence that the infection may be through CD4. In addition, we have found that infection of JAR and FD25 cells by HIV-1/Lai was enhanced in the presence of human antisera to HIV-1. This supports an additional role for immunoglobulin receptors (Fc-R) in the entry of virus into the cell. We report here evidence that CD4 and Fc-R on the cell surface play crucial roles in the entry of HIV into such placenta-derived cell lines.
Antibodies, Monoclonal↗
Analysis of human immunodeficiency virus type 1 integrase mutants.
The human immunodeficiency virus type-1 (HIV-1) integrase protein (IN) mediates the insertion of linear double-stranded viral DNA into the host genome. Mutations in IN can have different effects on the virus life cycle. In this study, Gag-Pol polyprotein processing, Tat synthesis, and viral replication were investigated in integrase-defective HIV-1 mutants. In the absence of IN synthesis, the Gag-Pol polyprotein stability, packaging, and/or processing was reduced. There was limited expression of Tat observed in IN mutants, but no viral replication.
Base Sequence↗
Inhibition of HIV-1 replication by cyclosporine A or related compounds correlates with the ability to disrupt the Gag-cyclophilin A interaction.
The HIV-1 Gag polyprotein specifically incorporates the cellular peptidylprolyl isomerase cyclophilin A into virions. HIV-1 replication is inhibited by cyclosporine A, an immunosuppressive drug which binds with high affinity to cyclophilin A and precludes interaction with the Gag polyprotein. Using a panel of four drugs, including cyclosporine A, two nonimmunosuppressive analogues of cyclosporine A which bind to cyclophilin A but which cannot form a tertiary complex with the calcium-dependent phosphatase calcineurin, and the structurally unrelated immunosuppressant FK506, we demonstrated that the antiviral effect of cyclosporine A is not due to blockade of calcineurin-mediated signal transduction pathways. Rather, the effectiveness of cyclosporine A and related compounds at inhibiting HIV-1 replication correlates with cyclophilin A-binding affinity and with the ability to disrupt the interaction between cyclophilin A and the HIV-1 Gag polyprotein. These results support the contention that the Gag-cyclophilin A interaction is required for HIV-1 replication.
Amino Acid Isomerases↗
Insights into the interaction between tRNA and primer binding site from characterization of a unique HIV-1 virus which stably maintains dual PBS complementary to tRNA(Gly) and tRNA(His).
Previously our laboratory constructed an HIV-1 which stably maintained a primer binding site (PBS) complementary to tRNA(His) by mutating the region of the provirus within U5 postulated to interact with the anticodon of tRNA(His) (J. Wakefield, S-M Kang, and C. D. Morrow, 1996, J. Virol, 70, 966-975). From the analysis of the virus obtained after long-term culture, we identified an unusual proviral DNA in which the U5-PBS region contained a dual PBS complementary to tRNA(Gly) and tRNA(His), respectively, separated by a 21-nucleotide intervening sequence. To determine if this U5-PBS region containing the dual PBS would give rise to an infectious virus, the mutant U5-PBS containing the dual PBS was subcloned into an infectious HIV-1 proviral clone, pHXB2; the resultant proviral DNA was designated as pHXB2(Gly-His). Transfection of pHXB2(Gly-His) into cells gave rise to infectious virus. Analysis of the U5-PBS region revealed that the virus stably maintained the dual PBS rather than revert back to the wild-type PBS. In addition to genomes with the PBS complementary to tRNA(Gly) and tRNA(His), proviral genomes were identified after extended in vitro culture which contained dual PBS complementary to tRNA(Gly) and tRNA(Phe). To determine which PBS could be used for reverse transcription, we utilized an endogenous reverse transcription/PCR method which could discriminate (based on molecular size of the products) between the minus strand DNA initiated from the two PBSs. The results of this assay demonstrated that either the PBS complementary to tRNA(Gly) or tRNA(His) could be used for the initiation of reverse transcription. The results of our study highlight the complex interrelationship between U5-PBS and primer tRNA required for positioning the tRNA at the PBS and provides new insights into how the tRNA primer used to initiate reverse transcription is selected.
Animals↗
Human immunodeficiency virus type 1 genetic evolution in patients with prolonged suppression of plasma viremia.
Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with combination drug regimens results in a reduction of plasma viral load to levels below the limit of detection. To investigate the genomic fluctuations in HIV-1 populations from long-term responders to antiviral therapies we analyzed the viral sequence evolution of env and pol genes from sequential peripheral blood mononuclear cell (PBMC) DNA samples of three infected patients. Analyses of sequences covering the V3 and flanking env regions obtained from blood samples at the beginning of the therapy and at 14 or 24 months from baseline revealed that HIV-1 quasispecies continue to evolve in the three patients following combination antiretroviral therapy. Minor drug-resistant mutant subpopulations were also searched for and found in one patient. Interestingly, no minor resistant subpopulations were found in the other two patients despite the fact that they showed evidence of ongoing viral replication. Finally, the genetic analysis of the env gene shows a reduction in PBMC env viral population diversity after long-term response to the therapy in all the patients analyzed.
Anti-HIV Agents↗
A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase.
Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of virus was reduced 100- to 1000-fold. By serial passage of our standard SV(STD) in Ae. albopictus cells in the presence of increasing concentrations of PZF, a mutant, SV(PZF), was derived, which was not inhibited by PZF. SV(PZF) is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease levels of UTP and CTP. Analysis of chimeric viruses containing sequences from the SV(PZF) and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region identified four mutations (nt 5750, 6627, 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T in nsP4. Characterization of viruses containing one or more of these mutations demonstrated that all three mutations in the nsP4 coding region are required to produce full resistance to PZF. Using a molecular model of nsP4 based on the structure of HIV reverse transcriptase, we located amino acid change M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these three amino acid changes in nsP4 alter the geometry of the NTP binding pocket so as to increase the affinity of the enzyme for CTP and UTP.
Adenosine↗