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N Yahi

Publications and source records attributed to N Yahi.

At least 19 recordsLinked to original sources
PubMed2001

Use of drug resistance sequence data for the systematic detection of non-B human immunodeficiency virus type 1 (HIV-1) subtypes: how to create a sentinel site for monitoring the genetic diversity of HIV-1 at a country scale.

To assess the molecular epidemiology of human immunodeficiency virus type 1 (HIV-1), a screening method was developed for identification of non-B subtypes from sequence data obtained for resistance testing. The method is based on the evaluation of the percentage of divergence of a given sequence from the reference B subtype HXB2. Analysis of 1720 reverse-transcriptase (RT) and 1824 protease sequences stored in a database allowed for the determination of a threshold level of divergence from HXB2 above which a non-B subtype could be unambiguously characterized regardless of the pattern of resistance mutations (>8.6% for RT; >10.8% for protease). This conclusion was validated by phylogenetic analysis of RT, protease, and env genes. Overall, 72 (4.2%) and 73 (4.0%) non-B sequences were identified in the RT and protease coding regions, respectively. This method allows for the rapid detection of non-B subtypes among retrospective, recent, and future RT and/or protease sequence databases.

Adolescent↗
PubMed2001

Genetic analysis of hiv type 1 strains in bujumbura (burundi): predominance of subtype c variant.

In order to characterize the HIV-1 strains circulating in Burundi, 18 blood samples from nontreated patients were collected in Bujumbura and viral DNA and RNA were sequenced in the env and pol genes, respectively. The phylogenetic analysis of the V3 coding region of HIV-1 gp120 revealed that 83% (15/18) of the isolates belonged to the C subtype. The RT and protease coding regions of the pol gene also clustered with subtype C. A potential A/C recombinant between the protease (subtype A) and the RT and V3 coding regions (both subtype C) was identified. Drug resistance mutations were not detected in the RT gene. However, mutation M36I, associated with resistance to ritonavir and nelfinavir, was found in 17 of 18 Burundi isolates. In conclusion, this first characterization of HIV-1 strains circulating in Burundi confirms the dramatic emergence of subtype C in East Africa.

Amino Acid Sequence↗
PubMed2001

Comparison of two commercial assays for the detection of insertion mutations of HIV-1 reverse transcriptase.

Insertions in the beta3-beta4 fingers subdomain of HIV-1 reverse transcriptase (RT) confer cross-resistance to various nucleoside analogs. The detection of these rearrangements in the region of codons 67-70 of RT is of primary importance for adapting and optimizing combination treatment regimen. Recent reports suggest that some genotyping techniques based on the hybridization of oligonucleotide probes may fail to detect insertion mutants of HIV-1 RT. In the present study, we have evaluated the efficiency of two commercial kits TruGene (based on Dye Primer sequencing) and Viroseq (Big Dye Terminator technique) for the detection of insertion mutations. The data were compared with an in-house dRhodamine sequencing method. Overall, all these cycle sequencing techniques were operative in the detection of insertion mutants. The best peak homogeneity in the electrophoregrams was observed with the Dye primer technique. However, specific compression artifacts were frequently encountered with this technique, rendering ambiguous the interpretation of the electrophoregrams in several regions of the sequence. This shortcoming did not occur with dRhodamine Dye terminator or Bigdye terminator cycle sequencing. In any case, a manual inspection of the electrophoregrams is highly recommended, for all types of cycle sequencing techniques, especially for detecting new mutational patterns of the RT and protease genes. Finally, some specific problems were encountered with the softwares provided with both Trugene and Viroseq kits.

Amino Acid Sequence↗
PubMed2000

Multidrug resistance genotypes (insertions in the beta3-beta4 finger subdomain and MDR mutations) of HIV-1 reverse transcriptase from extensively treated patients: incidence and association with other resistance mutations.

Multiple nucleoside resistance involves specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides. These include a cluster of five mutations in the reverse-transcriptase (RT) coding region (A62V, V75I, F77L, F116Y, and Q151M) generally referred to as multidrug resistance (MDR) mutations, and insertions of one or several amino acid residues between codons 67 and 70 of RT, a flexible region joining two antiparrallel beta sheets (beta3-beta4 insertions). The objectives of this study were (i) to determine the prevalence of multidrug resistance genotypes (MDR mutations and beta3-beta4 insertions) in a cohort of 632 patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral therapy, and (ii) to analyze the association of multidrug resistance genotypes with other resistance mutations in the RT and protease genes. Among viruses sequenced from these patients, 15 (2.4%) of them contained an insertion and 2 (0.3%) contained a deletion in the beta3-beta4 finger subdomain of RT. In 9 cases, the insertion was associated with a D67S, G, or E mutation. In addition, we identified 13 (2.1%) viruses harboring specific MDR mutations (mainly Q151M and/or A62V, V75I, F116Y). Interestingly, the A62V mutation was found in 6 of the 15 strains with an insertion, whereas the other MDR mutations were not observed in insertion mutant strains. Especially high levels of resistance to zidovudine were observed for viruses with a beta3-beta4 insertion in the background of A62V, L210W, and T215Y. Otherwise, MDR mutations and beta3-beta4 insertions were found in association with the classic mutations conferring resistance to zidovudine, lamivudine, nonnucleoside RT inhibitors, and protease inhibitors, according to treatment history. Finally, we observed a genome with a deletion of codon 70 associated with a Q151M MDR mutation. These data suggest that the emergence of HIV-1 multidrug resistance, which may occur in various genetic contexts, poses a challenging problem in formulating treatment strategies.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Prevalence of drug resistant mutants and virological response to combination therapy in patients with primary HIV-1 infection.

Baseline genotype resistance analysis was carried out in 48 adults with primary HIV-1 infection between 1995 and 1998 before starting early combination therapy. Seventeen percent (8/48) of the isolates displayed key mutations conferring resistance to reverse transcriptase (RT) inhibitors such as amino acid substitutions 215Y/F (5/48,10%), 70R (3/48, 6%), 184V (2%). Two percent (1/48) had a major mutation associated with resistance to protease inhibitors (D30N). Other mutations at positions 10, 15, 20, 33, 36, 46, 63, 71, 77, 82, 93 of the protease gene were frequent (73%). Among the 46 patients who were given antiretroviral combination therapy and who responded durably to treatment after 6 and 12 months, there was no significant difference between those harboring RT mutant strains (Group I) and those with wild-type isolates (Group II). No significant difference was found at months 6 and 12 between the two groups in terms of CD4+ cell counts. These findings suggest that the presence of drug-resistant strains at the time of primary HIV-1 infection does not necessarily predict drug failure. Other factors, such as adherence to treatment, tolerance and pharmacokinetics parameters are probably major determinants of virological response in patients with early therapeutic intervention.

Adult↗
PubMed1999

Genetic polymorphism near HIV-1 reverse transcriptase resistance-associated codons is a major obstacle for the line probe assay as an alternative method to sequence analysis.

The performance of the line probe assay (LIPA) for the detection of mutations conferring resistance to nucleoside inhibitors of HIV-1 reverse transcriptase was evaluated in comparison with sequence analysis. The tests were undertaken on plasma samples from 63 patients (61 receiving combination therapy and 2 without treatment at the time of inclusion). In 27 cases (43%) which included codons 41, 69, 70, 74, 184 and 215, the sequence of the RT gene was distinct from the hybridization probes used in LIPA. Correspondingly, LIPA gave uninterpretable results in 15, 30 and 41% of cases for codons 184, 215 and 41, respectively. Overall, the concordance between LIPA and sequence analysis varied from 52% (codons 41 and 215) to 85% (codon 70). These data show that the polymorphism of the nucleotide sequence near resistance-associated codons is a major shortcoming of LIPA.

Codon↗
PubMed1999

Mutation patterns of the reverse transcriptase and protease genes in human immunodeficiency virus type 1-infected patients undergoing combination therapy: survey of 787 sequences.

The aim of the present study was to evaluate the resistance-associated mutations in 302 human immunodeficiency virus type 1 (HIV-1)-infected patients receiving combination therapy and monitored in Marseille, France, hospitals from January 1997 to June 1998. In the reverse transcriptase (RT) gene, the most frequent mutations were found at codons 215 (53%), 41 (34%), and 67, 70, 184, and 210 (>20%). One deletion and two insertions in the beta3-beta4 hairpin loop of the finger subdomain (codon 69) were detected. Interesting associations and/or exclusions of specific mutations were observed. In 96% of RT genes, a mutation at codon 70 (most frequently, K70R) was associated with a wild-type genotype at position 210 (P < 10(-5)). Similarly, a mutation at codon 210 (most frequently, L210W) was generally associated with mutations at codons 41 (92%) and 215 (96%) but not at codon 219 (16%) or codon 70 (4%) (P < 10(-5)). In the protease gene, the most prevalent mutations were at codons 63 (84%), followed by codons 10, 36, 71, 77, and 93 (ca. 20%). As for RT, pairwise associations of mutations were observed. Analysis of the mutation patterns for patients with undetectable HIV-1 loads revealed a high proportion (65%) of wild-type RT genotypes but only 18% wild-type protease genotypes. For patients with high viral loads (>100,000 copies/ml), more than 50% of the RT and protease genes displayed three or more mutations. The significant correlation between the level of viremia in plasma and the number of resistance mutations in the protease (P = 0.007) and RT (P = 0.00078) genes strengthens the importance of defining the genotype of the predominant HIV-1 quasispecies before initiating antiretroviral therapy.

Amino Acid Substitution↗
PubMed1999

Comparison of human immunodeficiency virus type 1 (HIV-1) protease mutations in HIV-1 genomes detected in plasma and in peripheral blood mononuclear cells from patients receiving combination drug therapy.

Detections of mutations in the protease gene of human immunodeficiency virus type 1 in plasma and peripheral blood mononuclear cells (PBMC) were sought in two matched populations of 23 individuals receiving combination drug therapy with or without protease inhibitors. In the control group (23 patients not receiving protease inhibitors), no primary resistance mutations were found. In contrast, primary resistance mutations (especially at codons M46, V82, and L90) were found in 16 of 23 patients (70%) treated with protease inhibitors. In 30% of the cases, these mutations were detected in plasma but not in PBMC.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Human erythrocyte glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1 (HIV-1) entry: evidence for CD4-induced interactions between HIV-1 gp120 and reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3).

Glycosphingolipids from human erythrocytes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins. To identify the glycosphingolipid(s) which participates in the fusion process, we have analyzed the interaction of HIV-1 gp120 (X4 and R5X4 isolates) with reconstituted membrane microdomains of human erythrocyte glycosphingolipids. We identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main glycosphingolipids recognized by gp120. In the presence of CD4, Gb3 interacted preferentially with the X4 gp120, whereas GM3 interacted exclusively with the R5X4 gp120. These data suggest that glycosphingolipid microdomains are required in CD4-dependent fusion and that Gb3 and/or GM3 may function as alternative entry cofactors for selected HIV-1 isolates.

CD4 Antigens↗
PubMed1998

HIV-1-induced perturbations of glycosphingolipid metabolism are cell-specific and can be detected at early stages of HIV-1 infection.

The metabolism of glycosphingolipids (GSL) has been investigated in peripheral blood mononuclear cells (PBMC) from 8 patients at an early stage of HIV-1 infection. Following metabolic labeling of these cells with [14C]galactose, the GSL were purified and the radioactivity incorporated into each individual GSL quantitated by phosphoimaging. Compared with PBMC from seronegative donors, the GSL metabolism in PBMC from HIV-1-infected individuals was characterized by an increased synthesis of two GSL: the B-lymphocyte differentiation antigen globotriaosylceramide (Gb3, also referred to as CD77), and the monosialoganglioside GM3, a marker of T-lymphocytes and macrophages. The accumulation of Gb3 and GM3 in PBMC from HIV-1-infected patients was associated with the appearance of anti-Gb3 and anti-GM3 antibodies. Because these GSL are involved in the control of cell proliferation and signal transduction, such anti-GSL autoantibodies may contribute to the immune suppression during the course of HIV-1 infection. Studies on purified cell populations showed that GM3 accumulation occurred preferentially in HIV-1-infected monocytes/macrophages, whereas the synthesis glucosylceramide, the common precursor of complex GSL, was enhanced in both macrophages and CD4+ lymphocytes. Taken together, our data suggest that the dysregulation of GSL metabolism is an early event of HIV-1 pathogenesis that can induce important effects on immune cells homeostasis.

CD4-Positive T-Lymphocytes↗
PubMed1998

Stable rearrangements of the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase in plasma viruses from patients receiving combination therapy.

OBJECTIVES: To study the genetic rearrangements of HIV-1 reverse transcriptase (RT) in circulating viruses from patients under combination therapy, and to determine the impact of these changes on the virological response to treatment. METHODS: Blood samples were extracted from total RNA and amplified by RT-PCR. The HIV-1 RT and protease genes were sequenced by fluorescent dye terminator cycle sequencing. RESULTS: Specific rearrangements in the RT coding region (between amino acids 66 and 71) were documented in nine patients. This region, which corresponds to a loop between the beta3 and beta4 strands of the fingers subdomain of RT, is involved in the interaction between the enzyme and the template primer. In vitro data with recombinant enzymes have shown the importance of this domain in the processive polymerization of HIV-1 RT. The rearrangements (eight deletions/insertions and one deletion with conservation of the reading frame) did not affect the overall secondary structure of the fingers subdomain, as assessed by the Garnier Osguthorpe Robson prediction method. The changes were generally stable over a follow-up of 10-12 months. With the exception of two cases, most of the patients of this study did not respond efficiently to antiretroviral therapy as assessed by measurements of plasma viraemia. Correspondingly, the RT and protease genes sequenced from these patients displayed numerous resistance-associated mutations. CONCLUSION: Functional and stable rearrangements in the beta3-beta4 hairpin of HIV-1 RT can be found in circulating viruses from patients under combination therapy. These rearrangements may affect the virological response to antiretroviral therapy by increasing the processivity of RT, an enzymatic parameter that reflects the fidelity of the polymerization process.

Amino Acid Sequence↗
PubMed1998

Sulfatide inhibits HIV-1 entry into CD4-/CXCR4+ cells.

Sulfatide (3'sulfogalactosylceramide) is the natural sulfated derivative of galactosylceramide (GalCer), a glycosphingolipid receptor allowing HIV-1 infection of CD4-negative cells from neural and intestinal tissues. The incorporation of exogenous sulfatide into the plasma membrane of HT-29 (a CD4-/GalCer+/CXCR4+ human intestinal cell line) or RD (CD4-/GalCer-/ CXCR4+ human rhabdomyosarcoma) resulted in a dose-dependent inhibition of HIV-1 infection. Experiments with luciferase reporter viruses pseudotyped with HIV-1 or amphotropic murine leukemia virus envelopes demonstrated that sulfatide acts at the level of viral entry. Paradoxically, the transfer of sulfatide in the plasma membrane of various CD4- cells resulted in increased binding of HIV-1. Surface pressure measurements were conducted to study the interaction of gp120 with glycosphingolipid monolayers. The data showed that gp120 could penetrate into a monomolecular film of GalCer, confirming the role of this glycosphingolipid as a functional receptor for HIV-1. In contrast, the insertion of gp120 into a monolayer of sulfatide was very limited. Moreover, the incorporation of sulfatide in a monomolecular film of GalCer specifically inhibited the penetration of gp120. In conclusion, these data show that sulfatide mediates gp120 binding but, in marked contrast with GalCer, is not able to initiate the fusion event.

CD4 Antigens↗
PubMed1998

Sequential interaction of CD4 and HIV-1 gp120 with a reconstituted membrane patch of ganglioside GM3: implications for the role of glycolipids as potential HIV-1 fusion cofactors.

The fusion of HIV-1 with CD4+ cells involves, in addition to CD4, specific cell surface molecules acting as fusion cofactors. Recently, we reported that the V3 loop of HIV-1 gp120 binds to GM3, a ganglioside abundantly expressed on CD4+ lymphocytes and macrophages. In the present study, we show that CD4 interacts with a reconstituted patch of GM3 by measuring the surface pressure with a Langmuir film balance. A biphasic increase in surface pressure is observed after the sequential addition of CD4 and gp120 under the GM3 monolayer, indicating the formation of the trimolecular complex GM3-CD4-gp120. Neutralization of gp120 with an anti-V3 antibody inhibits the secondary interaction with GM3, suggesting that the CD4-induced conformational change in gp120 allows the V3 loop to interact with GM3. In conclusion, this study supports the concept that glycolipids can function as HIV-1 fusion cofactors.

Animals↗
PubMed1998

Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3.

Cellular glycosphingolipids mediate the fusion between some viruses and the plasma membrane of target cells. In the present study, we have analyzed the interaction of human immunodeficiency virus (HIV)-1 and HIV-2 surface envelope glycoproteins from distinct viral isolates with monolayers of various glycosphingolipids at the air-water interface. The penetration of the viral glycoproteins into glycosphingolipid monolayers was detected as an increase in the surface pressure. We found that HIV-1 recombinant gp120 (IIIB isolate) could penetrate into a monomolecular film of alpha-hydroxylated galactosylceramide (GalCer-HFA), while ceramides, GluCer, and nonhydroxylated GalCer were totally inactive. The glycoproteins isolated from HIV-1 isolates LAI and NDK and from HIV-2(ROD) could also interact with a GalCer-HFA monolayer, whereas gp120 from HIV-1(SEN) and HIV-1(89.6) did not react. These data correlated with the ability of the corresponding viruses to gain entry into the CD4(-)/GalCer+ cell line HT-29, demonstrating the determinant role of GalCer-HFA in this CD4-independent pathway of HIV-1 and HIV-2 infection. In contrast, all HIV-1 and HIV-2 glycoproteins tested were found to interact with a monolayer of GM3, a ganglioside abundantly expressed in the plasma membrane of CD4(+) lymphocytes and macrophages. A V3 loop-derived synthetic peptide inhibitor of HIV-1 and HIV-2 infection in both CD4(-) and CD4(+) cells could penetrate into various glycosphingolipid monolayers, including GalCer-HFA and GM3. Taken together, these data suggest that the adsorption of human immunodeficiency viruses to the surface of target cells involves an interaction between the V3 domain of the surface envelope glycoprotein and specific glycosphingolipids, i.e. GalCer-HFA for CD4(-) cells and GM3 for CD4(+) cells.

Amino Acid Sequence↗