Publications (9)61.32 Total impact
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Article: A non-infectious cell-based phenotypic assay for the assessment of HIV-1 susceptibility to protease inhibitors.
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ABSTRACT: HIV-1 genotyping is widely accepted as a diagnostic tool to optimize therapy changes in patients whose antiretroviral regimen is failing. Phenotyping can substantially complement the information obtained from genotyping, especially in the presence of complex mutational patterns. However, drug susceptibility tests are laborious and require biosafety facilities. We describe the molecular mechanism of a non-infectious HIV-1 protease phenotypic assay in eukaryotic cells and validate its applicability as a tool for monitoring drug resistance. A cloning vector containing the fusion protein green fluorescent protein-HIV-1 protease (GFP-PR) was modified to facilitate the insertion of HIV-1 protease from infected subjects. Real-time quantitative PCR and western blot analysis were used to establish the molecular mechanism of the new phenotypic assay. The method was validated by analysing HIV-1 protease from 46 clinical isolates. Statistical comparisons were made between values obtained using our assay and those reported from alternative standardized phenotypic assays. The capacity of HIV-1 protease to cleave cellular translation factors, such as the eukaryotic translation initiation factor 4 (eIF4GI) and the poly(A)-binding protein (PABP), led to cyclical accumulation of GFP that varied with the dose of protease inhibitors. Validation and comparison revealed a significant correlation with the Virco TYPE HIV-1 test (P < 0.0001, Spearman's ρ = 0.60), the Antivirogram test (P = 0.0001, Spearman's ρ = 0.60) and the Stanford HIVdb (P < 0.0001, Spearman's ρ = 0.69). This cell-based non-infectious phenotypic method with a well-understood molecular mechanism was highly reliable and comparable to other widely used assays. The method can be used for both phenotyping of HIV-1 viral isolates resistant to protease inhibitors and screening of new protease inhibitors.Journal of Antimicrobial Chemotherapy 01/2012; 67(1):32-8. · 5.07 Impact Factor -
Article: A376S in the connection subdomain of HIV-1 reverse transcriptase confers increased risk of virological failure to nevirapine therapy.
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ABSTRACT: The clinical relevance of mutations in the connection subdomain and the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) is uncertain. The risk of virological failure to nonnucleoside RT inhibitor (NNRTI)-based antiretroviral therapy (ART) was evaluated in NNRTI-naive patients who started NNRTIs in the EuroSIDA study after July 1997 according to preexisting substitutions in the connection subdomain and the RNase H domain of HIV-1 RT. An observed association between A376S and virological failure was further investigated by testing in vitro NNRTI susceptibility of single site-directed mutants and patient-derived recombinant viruses. Enzymatic assays also determined the effects of A376S on nevirapine and template-primer binding to HIV-1 RT. Virological failure occurred in 142 of 287 (49%) individuals: 77 receiving nevirapine (67%) and 65 receiving efavirenz (38%) (P < .001). Preexisting A376S was associated with an increased risk of virological failure to nevirapine (relative hazard [RH] = 10.4; 95% confidence interval [CI], 2.0-54.7), but it did not affect efavirenz outcome the same way (RH = 0.5; 95% CI, 0.1-2.2) (P value for interaction = .013). A376S conferred selective low-level nevirapine resistance in vitro, and led to greater affinity for double-stranded DNA. The A376S substitution in the connection subdomain of HIV-1 RT causes selective nevirapine resistance and confers an increased risk of virological failure to nevirapine-based ART.The Journal of Infectious Diseases 09/2011; 204(5):741-52. · 6.41 Impact Factor -
Article: Exosomes and retroviruses: the chicken or the egg?
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ABSTRACT: Retroviruses appropriate pre-existing cellular machineries to propagate. In the last decade, impressive similarities have been observed in the generation and dissemination in the host cells of retroviruses and small cellular vesicles known as exosomes. These cellular vesicles are thought to facilitate intercellular communication processes and mediate immune functions. However, their link to the retroviral life cycle has given rise to distinct hypotheses and puzzling dilemmas. Are exosomes the antecessors of retroviruses or do retroviruses merely exploit the same cellular machinery designated for exosome biosynthesis? Here, we address these fascinating evolutionary questions by reviewing recent discoveries and analysing the controversies surrounding them.Cellular Microbiology 10/2010; 13(1):10-7. · 5.46 Impact Factor -
Article: HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse?
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ABSTRACT: Exosomes are secreted cellular vesicles that can induce specific CD4(+) T cell responses in vivo when they interact with competent antigen-presenting cells like mature dendritic cells (mDCs). The Trojan exosome hypothesis proposes that retroviruses can take advantage of the cell-encoded intercellular vesicle traffic and exosome exchange pathway, moving between cells in the absence of fusion events in search of adequate target cells. Here, we discuss recent data supporting this hypothesis, which further explains how DCs can capture and internalize retroviruses like HIV-1 in the absence of fusion events, leading to the productive infection of interacting CD4(+) T cells and contributing to viral spread through a mechanism known as trans-infection. We suggest that HIV-1 can exploit an exosome antigen-dissemination pathway intrinsic to mDCs, allowing viral internalization and final trans-infection of CD4(+) T cells. In contrast to previous reports that focus on the ability of immature DCs to capture HIV in the mucosa, this review emphasizes the outstanding role that mature DCs could have promoting trans-infection in the lymph node, underscoring a new potential viral dissemination pathway.PLoS Pathogens 01/2010; 6(3):e1000740. · 9.13 Impact Factor -
Article: Effect of the human immunodeficiency virus type 1 reverse transcriptase polymorphism Leu-214 on replication capacity and drug susceptibility.
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ABSTRACT: A negative association between polymorphism Leu-214 and type-1 thymidine analogue mutations (TAM1) and a positive association with a clinically favorable virological response to thymidine analogue-based combination antiretroviral therapy have been described. In this study, the impact of Leu-214 on replication capacity and resistance to zidovudine (ZDV) of viruses containing TAM1 or TAM2 was determined. Leu-214 decreased the growth rate of viruses bearing Tyr-215, as well as their resistance to ZDV. This observation was confirmed by structural and molecular modeling data, suggesting a regulatory role for Leu-214 in the emergence and phenotypic resistance of TAM1.Journal of Virology 06/2009; 83(15):7434-9. · 5.40 Impact Factor -
Article: Contribution of immunological and virological factors to extremely severe primary HIV type 1 infection.
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ABSTRACT: During acute human immunodeficiency virus (HIV) infection, high viral loads and the induction of host immune responses typically coincide with the onset of clinical symptoms. However, clinically severe presentations during acute HIV type 1 (HIV-1) infection, including AIDS-defining symptoms, are unusual. Virus isolates were tested for clade, drug susceptibility, coreceptor use, and growth rate in 2 case reports of sexual transmission of HIV-1 infection. Human leukocyte antigen (HLA) genotype was determined, and HIV-1-specific cytotoxic T lymphocyte responses to an overlapping peptide set spanning the entire HIV clade A and clade B proteome were assayed. The viruses isolated in the 2 unrelated case reports of severe primary HIV-1 infection showed R5/X4 dual-mixed tropism, belonged to clade B and CRF02-AG, and were highly replicative in peripheral blood mononuclear cell culture. Impaired humoral responses were paralleled by a profound absence of HIV-1-specific cytotoxic T lymphocyte responses to the entire viral proteome in the 2 case reports. In 1 case report for which the virus source was available, there was a remarkable HLA similarity between the 2 patients involved in the transmission event, because 3 of 4 HLA-A and HLA-B alleles had matched HLA supertype for both patients. The data suggest that concurrence of viral and host factors contributes to the clinical severity of primary HIV-1 infection and that patients infected with highly replicative, dual-tropic viruses are more prone to develop AIDS-defining symptoms during acute infection if they are unable to mount humoral and cellular HIV-1-specific immune responses. The presence of concordant HLA supertypes might facilitate the preferential transmission of HLA-adapted viral variants, further accelerating disease progression.Clinical Infectious Diseases 01/2009; 48(2):229-38. · 9.15 Impact Factor -
Article: Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein.
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ABSTRACT: The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI(429-437) (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.Journal of Virology 11/2008; 83(2):1018-25. · 5.40 Impact Factor -
Article: Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway.
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ABSTRACT: Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.Blood 11/2008; 113(12):2732-41. · 9.90 Impact Factor -
Article: Relative fitness and replication capacity of a multinucleoside analogue-resistant clinical human immunodeficiency virus type 1 isolate with a deletion of codon 69 in the reverse transcriptase coding region.
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ABSTRACT: Deletions, insertions, and amino acid substitutions in the beta3-beta4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Delta69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Delta69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Delta69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Delta69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.Journal of Virology 06/2007; 81(9):4713-21. · 5.40 Impact Factor
Top co-authors
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Institutions
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2007–2012
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Hospital Universitari Germans Trias i Pujol
Badalona, Catalonia, Spain
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2011
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Irsicaixa - Institut de recerca de la SIDA
Badalona, Catalonia, Spain
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