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1.
Front Immunol ; 15: 1334236, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38444847

RESUMO

Introduction: Initiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population. Methods: Forty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested. Results: CA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study. Conclusions: Low CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.


Assuntos
Ácidos Nucleicos Livres , Infecções por HIV , Humanos , Adolescente , Criança , Estudos Transversais , RNA , Antirretrovirais/uso terapêutico , Citocinas , Infecções por HIV/tratamento farmacológico , DNA
2.
Viruses ; 15(9)2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37766218

RESUMO

Modern HIV-1 treatment effectively suppresses viral amplification in people living with HIV. However, the persistence of HIV-1 DNA as proviruses integrated into the human genome remains the main barrier to achieving a cure. Next-generation sequencing (NGS) offers increased sensitivity for characterising archived drug resistance mutations (DRMs) in HIV-1 DNA for improved treatment options. In this study, we present an ultra-sensitive targeted PCR assay coupled with NGS and a robust pipeline to characterise HIV-1 DNA DRMs from buffy coat samples. Our evaluation supports the use of this assay for Pan-HIV-1 analyses with reliable detection of DRMs across the HIV-1 Pol region. We propose this assay as a new valuable tool for monitoring archived HIV-1 drug resistance in virologically suppressed individuals, especially in clinical trials investigating novel therapeutic approaches.


Assuntos
Fármacos Anti-HIV , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Infecções por HIV/tratamento farmacológico , Genótipo , Farmacorresistência Viral/genética , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Antirretrovirais/uso terapêutico , Mutação , Sequenciamento de Nucleotídeos em Larga Escala
3.
Microbiol Spectr ; 10(3): e0024322, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35658711

RESUMO

Reliable and accurate quantification of cell-associated HIV DNA (CA HIV DNA) is critical for early infant diagnosis, clinical management of patients under therapy, and to inform new therapeutics efficacy. The present study assessed the variability of CA HIV DNA quantification obtained from various assays and the value of using reference materials to help harmonize the measurements. Using a common set of reagents, our multicenter collaborative study highlights significant variability of CA HIV DNA quantification and lower limit of quantification across assays. The quantification of CA HIV DNA from a panel of infected PBMCs can be harmonized through cross-subtype normalization but assay calibration with the commonly used 8E5 cell line failed to reduce quantification variability between assays, demonstrating the requirement to thoroughly evaluate reference material candidates to help improve the comparability of CA HIV DNA diagnostic assay performance. IMPORTANCE Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy. Our study demonstrates that HIV DNA measurement of the same samples can vary significantly from one laboratory to another, due to heterogeneity in the assay, protocol, and reagents used. We show that when carefully selected, reference materials can reduce measurement variability and harmonize HIV DNA quantification across laboratories, which will help contribute to improved diagnosis and clinical management of patients living with HIV.


Assuntos
Infecções por HIV , HIV-1 , DNA , DNA Viral/genética , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Laboratórios , Carga Viral/métodos
4.
Virol J ; 19(1): 77, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35501862

RESUMO

BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Criança , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA , SARS-CoV-2/genética
5.
Front Immunol ; 13: 875692, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35592310

RESUMO

Background: Identifying subphenotypes within heterogeneous diseases may have an impact in terms of therapeutic options. In this study, we aim to assess different subphenotypes in children living with human immunodeficiency virus (HIV-1), according to the clinical, virological, and immunological characteristics. Methods: We collected clinical and sociodemographic data, baseline viral load (VL), CD4 and CD8 count and percentage, age at initiation of ART, HIV DNA reservoir size in peripheral blood mononuclear cells (PBMCs), cell-associated RNA (CA-RNA), ultrasensitive VL, CD4 subsets (T effector CD25+, activated memory cells, Treg cells), humoral-specific HIV response (T-bet B cells), innate response (CD56dim natural killer (NK) cells, NKp46+, perforin), exhaustion markers (PD-1, PD-L1, DNAM), CD8 senescence, and biomarkers for T-lymphocyte thymic output (TREC) and endothelial activation (VCAM). The most informative variables were selected using an unsupervised lasso-type penalty selection for sparse clustering. Hierarchical clustering was performed using Pearson correlation as the distance metric and WARD.D2 as the clustering method. Internal validation was applied to select the best number of clusters. To compare the characteristics among clusters, boxplot and Kruskal Wallis test were assessed. Results: Three subphenotypes were discovered (cluster1: n=18, 45%; cluster2: n=11, 27.5%; cluster3: n=11, 27.5%). Patients in cluster1 were treated earlier, had higher baseline %CD4, low HIV reservoir size, low western blot score, higher TREC values, and lower VCAM values than the patients in the other clusters. In contrast, cluster3 was the less favorable. Patients were treated later and presented poorer outcomes with lower %CD4, and higher reservoir size, along with a higher percentage of CD8 immunosenescent cells, lower TREC, higher VCAM cytokine, and a higher %CD4 PD-1. Cluster2 was intermediate. Patients were like those of cluster1, but had lower levels of t-bet expression and higher HIV DNA reservoir size. Conclusions: Three HIV pediatric subphenotypes with different virological and immunological features were identified. The most favorable cluster was characterized by a higher rate of immune reconstitution and a slower disease progression, and the less favorable with more senescence and high reservoir size. In the near future therapeutic interventions for a path of a cure might be guided or supported by the different subphenotypes.


Assuntos
Infecções por HIV , HIV-1 , Criança , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/metabolismo , Receptor de Morte Celular Programada 1 , RNA
6.
Life (Basel) ; 13(1)2022 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-36676006

RESUMO

The dissemination of soil tares in the potato and sugar beet processing industry is one of the main paths for the spread of potato cyst nematodes (PCN), a severe quarantine pest. Efficient measures for the disinfestation of tare soil from PCN, but also from beet cyst nematodes (BCN), are needed. In our study, Globodera pallida (a PCN) and Heterodera schachtii (a BCN) cysts were sealed in gauze bags and imbedded in sedimentation basins. The cysts were either placed (a) in a presedimentation basin (Brukner basin) for three days, (b) in the presedimentation basin for three days and subsequently in sedimentation basins for nine weeks or (c) in sedimentation basins for nine weeks (without presedimentation). We tested the viability of the eggs and juveniles by hatching assays and using the reproduction rates in bioassays. We demonstrated that PCN and BCN imbedded in a sedimentation basin were only still showing some hatching activity after 2.5 weeks, while no hatching was observed when an additional Brukner basin treatment was conducted before sedimentation.

7.
J Int AIDS Soc ; 24(7): e25717, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34235857

RESUMO

INTRODUCTION: HIV infection causes pathological changes in the natural killer (NK) cell compartment that can be only partially restored by antiretroviral therapy (ART). We investigated NK cells phenotype and function in children with perinatally acquired HIV (PHIV) and long-term viral control (five years) due to effective ART in a multicentre cross-sectional European study (CARMA, EPIICAL consortium). The impact of age at ART start and viral reservoir was also evaluated. METHODS: Peripheral blood mononuclear cells (PBMCs) from 40 PHIV who started ART within two years of life (early treated patients (ET), ≤6 months; late treated patients (LT), > 6 months), with at least five years of HIV-1 suppression (<40 HIV copies/mL), were collected between November 2017 and August 2018. NK phenotype and function were analysed by flow cytometry and transcriptional profile of PBMCs by RNA-Seq. HIV-1 DNA was measured by real-time polymerase chain reaction (Data were analysed by Spearman correlation plots and multivariable Poisson regression model (adjusted for baseline %CD4 and RNA HIV viral load and for age at ART start as an interaction term, either ET or LT) to explore the association between NK cell parameters and HIV reservoir modulated by age at ART start. RESULTS: A significantly higher frequency of CD56neg NK cells was found in LT compared with ET. We further found in LT a positive correlation of CD56neg NK cells with HIV-1 DNA. LT also displayed increased expression of the NKG2D and NKp46 activating receptors and perforin compared with ET. Moreover, CD107a+ and IFN-γ+ frequencies in non-stimulated NK were associated with HIV-1 DNA in LT patients. Finally, RNA-Seq analysis showed in LT an up-regulation of genes related to NK-activating pathways and susceptibility to apoptosis compared with ET. CONCLUSIONS: We show that early initiation of ART during infancy preserves the NK compartment and is associated with lower HIV-1 reservoir. Such condition persists over adolescence due to long-term viral control achieved through effective ART.


Assuntos
Infecções por HIV , HIV-1 , Adolescente , Estudos Transversais , Infecções por HIV/tratamento farmacológico , Humanos , Lactente , Células Matadoras Naturais , Leucócitos Mononucleares
8.
Nat Microbiol ; 4(11): 1840-1850, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611641

RESUMO

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linfócitos T CD4-Positivos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Deleção de Genes , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Sinais de Localização Nuclear , Ligação Proteica , Conformação Proteica , RNA Viral/metabolismo , Desenvelopamento do Vírus , beta Carioferinas/genética
9.
PLoS Pathog ; 12(8): e1005774, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27509048

RESUMO

Elevated blood CXCL10/IP-10 levels during primary HIV-1 infection (PHI) were described as an independent marker of rapid disease onset, more robust than peak viremia or CD4 cell nadir. IP-10 enhances the recruitment of CXCR3+ cells, which include major HIV-target cells, raising the question if it promotes the establishment of viral reservoirs. We analyzed data from four cohorts of HIV+ patients, allowing us to study IP-10 levels before infection (Amsterdam cohort), as well as during controlled and uncontrolled viremia (ANRS cohorts). We also addressed IP-10 expression levels with regards to lymphoid tissues (LT) and blood viral reservoirs in patients and non-human primates. Pre-existing elevated IP-10 levels but not sCD63 associated with rapid CD4 T-cell loss upon HIV-1 infection. During PHI, IP-10 levels and to a lesser level IL-18 correlated with cell-associated HIV DNA, while 26 other inflammatory soluble markers did not. IP-10 levels tended to differ between HIV controllers with detectable and undetectable viremia. IP-10 was increased in SIV-exposed aviremic macaques with detectable SIV DNA in tissues. IP-10 mRNA was produced at higher levels in the small intestine than in colon or rectum. Jejunal IP-10+ cells corresponded to numerous small and round CD68neg cells as well as to macrophages. Blood IP-10 response negatively correlated with RORC (Th17 marker) gene expression in the small intestine. CXCR3 expression was higher on memory CD4+ T cells than any other immune cells. CD4 T cells from chronically infected animals expressed extremely high levels of intra-cellular CXCR3 suggesting internalization after ligand recognition. Elevated systemic IP-10 levels before infection associated with rapid disease progression. Systemic IP-10 during PHI correlated with HIV DNA. IP-10 production was regionalized in the intestine during early SIV infection and CD68+ and CD68neg haematopoietic cells in the small intestine appeared to be the major source of IP-10.


Assuntos
Quimiocina CXCL10/metabolismo , Infecções por HIV/imunologia , Intestino Delgado/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Separação Celular , Progressão da Doença , Citometria de Fluxo , HIV-1/imunologia , Humanos , Imuno-Histoquímica , Macaca , Reação em Cadeia da Polimerase , Vírus da Imunodeficiência Símia/imunologia , Carga Viral/imunologia , Viremia/imunologia
10.
J Virol Methods ; 202: 46-53, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24598230

RESUMO

Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at http://www.definetherain.org.uk. Optimisation of the analyses of raw ddPCR data using 'definetherain' indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach.


Assuntos
Biologia Computacional/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , HIV-1/genética , Humanos , Sensibilidade e Especificidade , Albumina Sérica/genética
11.
J Virol ; 88(4): 2025-34, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24307588

RESUMO

Macrophage infection is considered to play an important role in HIV-1 pathogenesis and persistence. Using a primary cell-based coculture model, we show that monocyte-derived macrophages (MDM) efficiently transmit a high-multiplicity HIV-1 infection to autologous CD4(+) T cells through a viral envelope glycoprotein (Env) receptor- and actin-dependent virological synapse (VS), facilitated by interactions between ICAM-1 and LFA-1. Virological synapse (VS)-mediated transmission by MDM results in high levels of T cell HIV-1 integration and is 1 to 2 orders of magnitude more efficient than cell-free infection. This mode of cell-to-cell transmission is broadly susceptible to the activity of CD4 binding site (CD4bs) and glycan or glycopeptide epitope-specific broadly neutralizing monoclonal antibodies (bNMAbs) but shows resistance to bNMAbs targeting the Env gp41 subunit membrane-proximal external region (MPER). These data define for the first time the structure and function of the macrophage-to-T cell VS and have important implications for bNMAb activity in HIV-1 prophylaxis and therapy. IMPORTANCE The ability of HIV-1 to move directly between contacting immune cells allows efficient viral dissemination with the potential to evade antibody attack. Here, we show that HIV-1 spreads from infected macrophages to T cells via a structure called a virological synapse that maintains extended contact between the two cell types, allowing transfer of multiple infectious events to the T cell. This process allows the virus to avoid neutralization by a class of antibody targeting the gp41 subunit of the envelope glycoproteins. These results have implications for viral spread in vivo and the specificities of neutralizing antibody elicited by antibody-based vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/transmissão , Evasão da Resposta Imune/imunologia , Sinapses Imunológicas/virologia , Macrófagos/imunologia , Análise de Variância , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/virologia , Primers do DNA/genética , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Luciferases , Antígeno-1 Associado à Função Linfocitária/metabolismo , Macrófagos/virologia , Microscopia Confocal , Testes de Neutralização , Reação em Cadeia da Polimerase , Imagem com Lapso de Tempo
12.
Blood ; 121(6): 951-61, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-23255555

RESUMO

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.


Assuntos
Infecções por HIV/imunologia , Imunidade nas Mucosas/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Estudos de Coortes , Escherichia coli/imunologia , Feminino , Citometria de Fluxo , HIV/efeitos dos fármacos , HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Imuno-Histoquímica , Interleucina-17/imunologia , Interleucina-17/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CCR6/imunologia , Receptores CCR6/metabolismo , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Fatores de Tempo
13.
Retrovirology ; 6: 32, 2009 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-19348676

RESUMO

BACKGROUND: Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed. RESULTS: We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 x 10-4 per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo error rate of 1.1 x 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector. CONCLUSION: Our results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.


Assuntos
Genoma Viral/genética , Spumavirus/genética , Desaminases APOBEC , Linhagem Celular , Citidina Desaminase , Citosina Desaminase/metabolismo , Humanos , Mutação , Proteínas dos Retroviridae/metabolismo , Moldes Genéticos , Replicação Viral
14.
Virology ; 373(1): 51-60, 2008 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-18078974

RESUMO

Similar to the lentiviruses family of retroviruses, foamy viruses (FVs) contain purine-rich sequences located in the center of the genome. Their function on viral replication or vector transfer remains elusive, although dual initiation of plus-strand reverse transcription has been suggested. To elucidate the physical nature of the central region of the prototype FV (PFV) genome, we performed 3' and 5' RACE experiments. Our results revealed that the PFV genome contains a centrally located gap in the DNA plus-strand with no definite termination and start point and of variable length. We did not find evidence for a DNA flap region. The PFV isolate harbors four centrally located purine-rich elements (A-D). Only the D element is identical in sequence to the 3' poly purine tract (PPT). We mutated these elements while conserving or altering the overlapping pol reading frame and analyzed the mutants for transient replication in an infectious or for vector transfer in a replication-deficient background. In addition, we determined the protein composition of the respective viral particles. The A and B elements appeared to play a role in Pol protein encapsidation, the C element is likely involved in regulating gene expression, while mutation of the D element resulted in an insignificant reduction in transiently replicating virus and an approximately 50% reduction in vector titer. The reason for this deficit remains to be elucidated.


Assuntos
Purinas/química , Spumavirus/genética , Replicação Viral , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , DNA Complementar/genética , DNA Complementar/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene pol/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Poli A/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Transcrição Reversa , Spumavirus/química , Spumavirus/fisiologia
15.
Virology ; 370(1): 151-7, 2008 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-17904181

RESUMO

Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.


Assuntos
Farmacorresistência Viral/genética , Genes pol/genética , Mutação , Inibidores da Transcriptase Reversa/farmacologia , Vírus Espumoso dos Símios/efeitos dos fármacos , Zidovudina/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Produtos do Gene pol/química , Produtos do Gene pol/genética , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Vírus Espumoso dos Símios/genética , Vírus Espumoso dos Símios/crescimento & desenvolvimento , Replicação Viral
16.
J Virol ; 78(5): 2472-7, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14963145

RESUMO

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5' end of the U3 region in the 3' LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.


Assuntos
Spumavirus/fisiologia , Integração Viral , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Vetores Genéticos/genética , Humanos , Mutação/genética , Provírus/genética , Provírus/fisiologia , Recombinação Genética/genética , Spumavirus/genética , Sequências Repetidas Terminais/genética , Integração Viral/genética , Replicação Viral
17.
J Virol ; 77(21): 11324-31, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14557618

RESUMO

Crucial aspects of the foamy virus (FV) replication strategy have so far only been investigated for the prototypic FV (PFV) isolate, which is supposed to be derived from nonhuman primates. To study whether the unusual features of this replication pathway also apply to more-distantly related FVs, we constructed feline FV (FFV) infectious molecular clones and vectors. It is shown by quantitative RNA and DNA PCR analysis that FFV virions contain more RNA than DNA. Full-length linear DNA was found in extracellular FFV by Southern blot analysis. Similar to PFV, azidothymidine inhibition experiments and the transfection of nucleic acids extracted from extracellular FFV indicated that DNA is the functional relevant FFV genome. Unlike PFV, no evidence was found indicating that FFV recycles its DNA into the nucleus.


Assuntos
Genoma Viral , Spumavirus/fisiologia , Replicação Viral , Animais , Gatos , Linhagem Celular , Cricetinae , DNA Viral/genética , DNA Viral/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Spumavirus/genética , Spumavirus/patogenicidade , Transfecção , Vírion/metabolismo , Vírion/patogenicidade
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