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PubMed1998

The potential roles of 4-1BB costimulation in HIV type 1 infection.

The potential role of 4-1BB in human immunodeficiency virus (HIV-1)-infected T cells was investigated with HIV-1-infected subjects. 4-1BB expression was readily inducible on PHA stimulation of T cells from HIV-1-infected individuals. The level of 4-1BB expression and the percentage of 4-1BB-expressing T cells were higher in HIV-1+ individuals than in the HIV-1- controls (p < 0.01). The difference in 4-1BB expression was more significant in CD8+ T cells and the high level of 4-1BB expression was correlated with low CD4+ T cell counts (r = -0.63, p < 0.05). 4-1BB signal cooperated with CD28 for proper HIV-1+ CD4+ T cell proliferation. In addition, cross-linking 4-1BB with agonistic monoclonal antibody enhanced HIV-1 replication both in primary stimulation and secondary restimulation of CD4+ T cells from HIV-1+ individuals. To test whether 4-1BB cross-linking signals an activation of HIV-1, J8-1, a 4-1BB+ Jurkat subline, was transiently transfected with pHIV-1-LTR-CAT plasmid and stimulated through 4-1BB. Combined stimulation of 4-1BB and CD3 resulted in an enhanced CAT activity compared with CD3 stimulation alone. Thus, 4-1BB may be involved in the activation of HIV-1 replication from latently infected CD4+ T cells.

Antibodies, Monoclonal↗
PubMed2004

Failure to detect human immunodeficiency virus type 1 superinfection in 28 HIV-seroconcordant individuals with high risk of reexposure to the virus.

We have examined the occurrence of HIV-1 superinfection in 14 HIV-seroconcordant couples (i.e., partners were independently infected with different HIV-1 strains) with high risk of reexposure to the virus. Phylogenetic analyses based on pol and env global sequences obtained from more than 100 longitudinal plasma samples (corresponding to a period of 1-4 years) failed to detect HIV-1 superinfection in this cohort of patients. Our results suggest that despite recent reports of HIV-1 reinfection, chronic HIV infection seems to confer protection against superinfection with a second HIV-1 strain.

CD4 Lymphocyte Count↗
PubMed2004

Inhibition of HIV type 1 replication using lentiviral-mediated delivery of mutant tRNA(Lys3)A58U.

In previous studies, we showed that residue A58 of cellular tRNALys3 is necessary for appropriate termination of viral plus-strand strong-stop DNA (+SS DNA), and therefore plays a critical role in the life cycle of HIV-1. We also performed proof-of-principle studies that established that a mutant form of this tRNA primer (tRNA(Lys3)A58U, which lacks the M1A58 residue necessary for +SS DNA termination) could inhibit HIV-1 replication. In the present work, we examined whether a third generation lentiviral vector (SIN) could be used to deliver tRNA(Lys3)A58U to CEM cells. Using both viral kinetic studies and limiting dilution assays (LDA), we observed significant impairment of HIV-1 replication, up to 3 logs in the LDA, in CEM sublines expressing mutant tRNA(Lys3)A58U. No inhibition occurred in cells that either expressed wild-type tRNA(Lys3) or were transduced with empty SIN vector. Further, we observed impairment of viral replication using primary isolates of both HIV-1 and HIV-2 in sublines containing tRNA(Lys3)A58U. We also detected "breakthrough" HIV-1 replication in some tRNA(Lys3)A58U-expressing cultures. Interestingly, analyzed breakthrough viruses appeared to be both genetically and phenotypically wild type. One possible explanation for virological breakthrough is that it reflects the gradual accumulation of HIV-1 within the infected cell culture, to a level that ultimately exceeds the containment "threshold" conferred by tRNA(Lys3)A58U. The fact that HIV-1 does not appear to acquire heritable resistance to tRNA(Lys3)A58U-mediated blockade differentiates this antiviral modality from other therapeutic interventions. It also suggests that tRNA-mediated inhibition of viral replication might be a valuable adjunct to other antiviral approaches.

Cell Line↗
PubMed2005

Molecular epidemiology of the heterosexual HIV-1 transmission in Kunming, Yunnan Province of China suggests origin from the local IDU epidemic.

Molecular epidemiological investigation was conducted among injecting drug users (IDUs) (n = 11) and heterosexuals (n = 15) in Kunming, Yunnan Province of China. HIV-1 genotypes were determined based on the nucleotide sequences of 2.6-kb gag-RT region. The distribution of genotypes among IDUs was as follows: CRF07_BC (5/11) and CRF08_BC (5/11); subtype B' (1/11). Similarly, a majority of Kunming heterosexuals (14/15) were infected with CRF07_BC (4/15), CRF08_BC (6 /15), or subtype B' (4/15), known to predominate among IDUs in China. This contrasts with trends in the coastal regions of China and surrounding southeastern Asian countries, where CRF01_AE predominates among heterosexuals. The heterosexual HIV-1 epidemic in Kunming thus appears to derive from the local IDU epidemic. Of note, subtype B' was the most prevalent strain among heterosexuals before 1997, while CRF07_BC and CRF08_BC became predominant in 2002, indicating a transition of HIV-1 genotype distribution between the early and the more recent samples from Kunming heterosexuals.

China↗
PubMed2006

Short partially double-stranded oligodeoxynucleotide induces reverse transcriptase/RNase H-mediated cleavage of HIV RNA and contributes to abrogation of infectivity of virions.

We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) directly in HIV virions. The ODN A consists of an antisense and a passenger strand, and was designed to target the polyp-urine tract (PPT) of HIV-1, a conserved region of the viral genome. It leads to HIV reverse transcriptase/ribonuclease H (RT/RNase H)-dependent degradation of the RNA in viral particles. Illimaquinone, a specific inhibitor of RNase H, activity of HIV RT/RNase H, prevents RNA cleavage. The effect of the ODN A is sequence-specific and the passenger strand is important, since a lack or alteration of this strand reduces the antiviral activity of the ODN. ODN A has a stronger antiviral effect compared to a control ODN CO, targeted to a site outside of the PPT. The pretreatment with ODN A strongly reduced the infectivity of virions in cell culture in the absence of any DNA carriers or detergents.

Antiviral Agents↗
PubMed2006

High diversity of HIV type 1 in Algeria.

We have sequenced different genes of HIV-1 strains from infected individuals recruited in various geographic parts of Algeria; phylogenetic trees were constructed yielding molecular characterization of these strains. Subtype B accounts for 56% of the samples studied and is therefore the predominant subtype, particularly in the north part of the country; but there is a high diversity of the virus including CRF02_AG, CRF06_cpx, CRF02/CRF06 interrecombinants, and different other intersubtype and/or inter-CRF recombinants. The prevalence of these non-B viruses increases in the south part of Algeria that borders sub-Saharan African countries. The high diversity of HIV-1 in Algeria has implications for virological follow-up, resistance surveys, and vaccine design.

Adolescent↗
PubMed1992

Poly(L-lysine)-conjugated oligonucleotides promote sequence-specific inhibition of acute HIV-1 infection.

Previously, we have reported that conjugation of antisense oligonucleotides to poly(L-lysine) (PLL) lowers their inhibitory concentration in several biological models. We have now tested these conjugates for inhibition of human immunodeficiency virus type 1 (HIV-1) replication. PLL-conjugated oligonucleotides complementary to the translation initiation site of Tat protein protect cells from the cytopathic effect of HIV-1 in acute infection assays. The EC50 of conjugates is approximately 0.15 microM, which represents a strong reduction in concentration as compared to nonconjugated oligonucleotides (EC50 = 20 microM). In contrast with most reports in the literature, we have observed sequence specific antiviral effects with PLL conjugates. This was particularly noteworthy in antiviral experiments performed with HIV-1 isolates presenting heterogeneity in the 5' end of the tat mRNA sequence. Two mismatches at the target site were sufficient to reduce very significantly the antiviral activity of the conjugates but did not modify the effect of nonconjugated oligonucleotides. Unlike free oligonucleotides, PLL-conjugated ones do not interfere with virus penetration and/or reverse transcription as demonstrated by polymerase chain reaction (PCR) analysis of viral DNA.

Animals↗
PubMed2005

Recognition patterns of HLA-A2-restricted human immunodeficiency virus-1-specific cytotoxic T-lymphocytes in a cohort of HIV-1-infected individuals.

The cytotoxic T-lymphocytes (CTL) response to three histocompatibility leukocyte antigen (HLA)-A2-restricted CTL epitopes was investigated in a cohort of 51 HLA-A2-positive human immunodeficiency-1 (HIV-1)-infected subjects. CTL activity was evaluated by testing peptide stimulated peripheral blood mononuclear cells (PBMC) in chromium release assays. The most prevalent CTL response was directed to the RT-peptide ILKEPVHGV (IV9) recognized by 37.3%. The p17-peptide SLYNTVATL (SL9), reported to be the immunodominant epitope in chronically infected untreated patients, was recognized only by 13.7%. Only 9.8% recognized both IV9 and SL9, and none recognized the RT-peptide VIYQYMDDL (VL9). CTL activity correlated significantly with absolute CD8 T-cell counts but not with CD4 counts, viral load, or antiviral therapy. Analysis of the recognition patterns of amino acid substitutions in the IV9 epitope revealed the presence of at least four functionally different T-cell receptors (TCR) in this cohort. All analyzed mutations within the TCR recognition site of this epitope could abrogate CTL recognition by individual CTL clones, but all were fully immunogenic for other CTL clones with peptide-sensitizing capacities similar to that of IV9. Further studies should be performed to evaluate whether a convergent epitope vaccination strategy using immunogenic variants of CTL epitopes is a feasible approach to broaden the TCR repertoire and to inhibit CTL escape.

Anti-HIV Agents↗
PubMed1995

DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene.

A 25mer oligonucleotide containing a single N-(deoxyguanosin-8-yl-)-1-aminopyrene (dGAP), the major DNA adduct formed by reductively activated 1-nitropyrene, was synthesized. The adduct was located at nucleotide 21 from the 3' end. DNA synthesis on this template by human DNA polymerases alpha and beta, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site. Only when a 3'-->5' exonuclease-deficient Klenow fragment was used was incorporation of a nucleotide opposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent. Only a relatively small proportion of full-length product (< 5%) was detected. In the presence of Mn2+, the efficiency of bypass with this polymerase increased. When a 20mer primer was elongated in the presence of only one nucleotide triphosphate, deoxycytidylic acid was preferentially incorporated opposite the adduct. Deoxycytidine opposite the adduct was also preferred when a set of 21mer primers (containing each of the four nucleotides opposite dGAP) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these results, extension of a 15mer primer was carried out with all four deoxynucleotide triphosphates and the products were isolated. Maxam--Gilbert sequencing of each elongation product showed that primer extension occurred in an error-free manner. We conclude that dGAP is a strong block of DNA replication. However, when translesion synthesis occurs, it is largely accurate.

Base Sequence↗
PubMed1998

Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.

Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway.

CD4 Antigens↗
PubMed2003

Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7.

Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses, can infect non-dividing cells. This property depends on the active nuclear import of its intracellular reverse transcription complex (RTC). We have studied nuclear import of purified HIV-1 RTCs in primary macrophages and found that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in the process. Nuclear import of RTCs requires, in addition, energy and the components of the Ran system. Depletion of importin 7 from cultured cells by small interfering RNA inhibits HIV-1 infection. These results provide a new insight into the molecular mechanism for HIV-1 nuclear import and reveal potential targets for therapeutic intervention.

Active Transport, Cell Nucleus↗
PubMed1995

Human immunodeficiency virus and Mycobacterium avium complex coinfection of monocytoid cells results in reciprocal enhancement of multiplication.

Disseminated Mycobacterium avium complex (MAC) infection is an important opportunistic infection in AIDS patients. Because cells of macrophage lineage are targets for both human immunodeficiency virus (HIV) and MAC, the monocytoid cell line U937 was coinfected with both pathogens. Coinfected cultures had increased HIV replication (more than threefold at day 6) and an increased percentage of HIV-infected cells compared with cultures infected only with HIV. The kinetics of HIV replication were significantly increased in this coinfection system as measured by flow cytometry. When cells were infected concurrently, the rate of intracellular growth of MAC was not significantly affected. However, cells preinfected with HIV before infection with MAC showed significant enhancement of MAC growth compared with control cells. The kinetics of cell death were also increased in the coinfection system compared with singly infected controls. Thus, coinfection of monocytoid cells with HIV and MAC in vitro results in reciprocal enhancement of multiplication.

Cell Survival↗
PubMed1995

Ex vivo expansion of CD4 lymphocytes from human immunodeficiency virus type 1-infected persons in the presence of combination antiretroviral agents.

Expansion of CD4 lymphocytes from human immunodeficiency virus type 1 (HIV-1)-infected persons ex vivo has been limited by enhanced virus replication and cell death. The successful expansion of functional CD4 lymphocytes from HIV-1-infected persons has now been accomplished using a bispecific monoclonal antibody to CD3 and CD8 in combination with three antiretroviral agents. CD4 lymphocytes were polyclonally expanded by a factor of 10(3)-10(7) during 4-8 weeks in culture. Supernatants from most cultures were persistently HIV-1 p24 antigen-negative by day 14 and remained negative despite removal of antiretroviral agents at day 28. In such cultures, HIV-1 could not be recovered by cocultivation, and amounts of HIV-1 DNA declined or remained stable at low levels, eventually becoming undetectable in 2 cases. This approach establishes the feasibility of CD4 lymphocyte expansion in persons with HIV disease and may be useful for immune-based or gene therapies.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Oligoclonal CD8 lymphocytes from persons with asymptomatic human immunodeficiency virus (HIV) type 1 infection inhibit HIV-1 replication.

CD8 lymphocytes from asymptomatic human immunodeficiency virus (HIV) type 1-infected patients can suppress virus production from infected CD4 cells. Suppressive activity is separate and distinct from cytotoxic T lymphocyte (CTL) reactivities and is likely mediated by a soluble factor(s). The majority of HIV-1 suppression studies have been done in the context of bulk CD8 cell cultures. In this study, viral suppression was characterized by clonal populations of CD8 cells derived from HIV-1-infected patients. Most of the suppressive clones were devoid of detectable CTL reactivity against env-, gag-, pol-, and nef-expressing targets. Among the suppressive clones derived from an individual patient, a marked heterogeneity was evident with respect to phenotypic markers, cytokine production, and T cell receptor V beta expression. These results suggest that noncytolytic virus suppression is oligoclonal in nature. Clones provide tools for future studies aimed at understanding the mechanism of suppression and identifying the suppressive factor.

CD8-Positive T-Lymphocytes↗
PubMed1995

A mAb against HLA-A2 can be influenced both positively and negatively by the associated peptide.

Presentation of peptides by class I HLA molecules is essential for the development of a T cell-mediated immune response. TCRs have the ability to discriminate among large numbers of different HLA-peptide complexes. We have identified a mAb, MA2.1, that also discriminates among HLA-A2 associated with different peptides. A soluble form of HLA-A2 bound to single peptides was prepared and its serological reactivity was studied using four mAbs. Three antibodies, W6/32, BB7.2 and BBM.1, recognized all the complexes equally. MA2.1, however, recognized most of the complexes equally but showed markedly different reactivity to two peptides bound to HLA-A2. MA2.1 recognized HLA-A2 complexed with the HIV-1 p17 epitope (SLYNTVATL) at least 30 times more strongly than all other complexes studied and this enhanced reactivity was found to be sensitive to a point mutation of threonine to alanine at position 8 in the peptide. In addition, MA2.1 had a very low reactivity for HLA-A2 complexed with the peptide TLWVDPYEV. Previous studies have mapped the binding epitope of MA2.1 to the alpha 1 and alpha 2 helices of HLA-A2, suggesting two possible explanations for the ability of the bound peptide to influence MA2.1 reactivity. Either MA2.1 is sensitive to peptide-induced conformational changes of the helices, or it directly contacts certain peptides in the groove of HLA-A2.

Amino Acid Sequence↗
PubMed1997

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes.

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.

Antigen Presentation↗
PubMed1994

Mechanism of inhibition of HIV-1 infection in vitro by guanine-rich oligonucleotides modified at the 5' terminal by dimethoxytrityl residue.

Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA-1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction.

Antiviral Agents↗
PubMed2000

In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase.

Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.

Animals↗

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