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The immunological role of cell wall components from diverse Mycobacterium tuberculosis clinical isolates in regulating HIV-1 replication in human macrophagesNdengane, Mthawelanga 11 September 2023 (has links) (PDF)
Human immunodeficiency virus type 1 (HIV-1) and Mycobacterium tuberculosis (Mtb) coinfection remains a major global health threat. Both pathogens synergistically drive pathogenesis of the other. The risk of developing active tuberculosis (TB) is increased in people living with HIV-1, even in those receiving antiretroviral therapy (ART), whilst TB was responsible for 15 % of HIV-related deaths in 2020. Mtb co-infection increases the likelihood of transcriptionally activating HIV-1 replication potentially due to bioactive Mtb lipids engaging macrophage surface receptors, thus triggering signaling pathways which activate human transcriptional factors (hTF) and production of inflammatory cytokines capable of activating HIV-1 transcription. This work investigated the hypothesis that clinical Mtb strains with single nucleotide polymorphisms (SNP) in lipid-metabolizing genes, required for cell wall lipid biosynthesis, differentially affect HIV-1 replication and human macrophage inflammatory response during Mtb-HIV-1 co-infection in vitro. Monocyte derived macrophages (MDM) were the predominant model used to investigate this phenomenon. Infections, in the presence or absence of HIV-1 co-infection, were performed using either lineage 2 or lineage 4 clinical strains with non-synonymous SNP in polyketide synthase 2 (pks2) required for sulfolipid 1 (SL-1) biosynthesis and compared to control infections using phylogenetically close clinical strains without the SNP of interest and canonical lineage 2 and 4 laboratory strains (H37RvP1939/T605, CDC1551WT and HN878WT). Secreted cytokines and chemokines were measured in supernatant (SN) by Luminex. The effect of Mtb on HIV-1 viral production was assessed by measuring HIV-1 Gag p24 in the SN of co-infected MDM or SN of HIV-1 infected MDM incubated with conditioned media from Mtb-infected MDM. The influence of Mtb on HIV-1 transcriptional activity was measured using a transgenic cell line (TZM-bl) with Luciferase reporter under HIV-1 long terminal repeat (LTR) expression. The impact of incubating TZM-bl cells in Mtb-induced conditioned media before or after HIV-1 infection was assessed. One pair of phylogenetically close clinical strains with and without a pks2 SNP of interest (EX30Q1939/A605 and MRC16P1939/A605) with interesting lipid and inflammatory phenotypes, and H37RvP1939/T605 as a lineage 4 control, were subject to single nucleotide mutagenesis using recombineering to either revert SNP of interest to match the alleles of H37Rv or introduce the SNP of interest into the control strains. The wild-type and mutant strains were used in a trans-well assay to infect MDM in the presence of HIV-1 co-infection in the top chamber, while simultaneously mimicking the bystander effect of cytokine-mediated HIV-1 regulation in the bottom chamber which was only infected with HIV-1. Results demonstrate there was increased cytokine production by MDM infected with MRC16P1939/A605 in both the presence and absence of HIV-1 co-infection compared to its phylogenetically close paired strain EX30 Q1939/A605. The data shows that there was no difference in LTR activity in TZM-bl cells co-incubated with inflammatory environment between the strains of interests, however co-incubation of TZM-bl cells with Mtb-induced inflammatory environment generally increased LTR activity during HIV-1; a proxy for HIV-1 replication. In the trans-well co-infection assay, a significant positive association between production of HIVp24 and secretion of CCL2 was observed, whilst IL-1β secretion showed a significant negative relationship with the production of HIVp24, with donor variability in baseline cytokine production also associated with the extent of HIVp24, CCL2, IL-1β and IL-8 production. Introduction of the pks2 T605A SNP into H37RvP1939/T605 and reversion in EX30Q1939/A605T significantly modified their inflammatory phenotype. Together these results support the hypothesis that Mtb clinical stains with genetic variation in cell wall lipid biosynthesis impacts the inflammatory milieu and, subsequently, HIV-1 replication during co-infection. The outcome of Mtb-HIV co-infection is therefore not homogenous but contingent on the phenotype of infecting Mtb strain and individual.
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Effects of traditional Chinese medicinal herbal extracts on HIV-1 replicationWang, Ting 16 March 2011 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Background: The current treatment for HIV/AIDS is called highly active antiretroviral therapy (HAART) and is a combination of anti-HIV reverse transcriptase inhibitors and protease inhibitors. HAART is capable of suppressing HIV replication and subsequently improving the patients’ survival. However, the issues associated with use of HARRT such as the high cost, severe side-effects, and drug resistance have called for development of alternative anti-HIV therapeutic strategies. In this study, we screened several traditional Chinese medicinal herbal extracts for their anti-HIV activities and determined their anti-HIV mechanisms.
Methods: Nine traditional Chinese medicinal (TCM) herbal plants and their respective parts derived from Hainan Island, China were extracted using a series of organic solvents, vacuum dried, and dissolved in dimethyl sulfoxide. Initial anti-HIV activity and cytotoxicity of these extracts were evaluated in HIV-infected human CD4+ T lymphocytes Jurkat. Extracts of higher anti-HIV activities and lower cytotoxicity were selected from the initial screening, and further examined for their effects on HIV-1 entry, post-entry, reverse transcriptase, gene transcription and expression using combined virology, cell biology and biochemistry techniques.
Results: Four extracts derived from two different herbal plants completely blocked HIV-1 replication and showed little cytotoxicity at a concentration of 10 g/ml. None of these four extracts had any inhibitory effects on HIV-1 long terminal repeat promoter. Two of them exhibited direct inhibitory activity against HIV-1 reverse transcriptase (RT). All four extracts showed significant blocking of HIV-1 entry into target cells.
Conclusions: These results demonstrated that four TCM extracts were capable of preventing HIV-1 infection and replication by blocking viral entry and/or directly inhibiting the RT activity. These results suggest the possibility of developing these extracts as potential anti-HIV therapeutic agents.
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Nuclear Trafficking of the HIV-1 Pre-Integration Complex Depends on the ADAM10 Intracellular DomainEndsley, Mark A., Somasunderam, Anoma D., Li, Guangyu, Oezguen, Numan, Thiviyanathan, Varatharasa, Murray, James L., Rubin, Donald H., Hodge, Thomas W., O'Brien, William A., Lewis, Briana, Ferguson, Monique R. 01 April 2014 (has links)
Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4+ T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry.
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Phenotypic and functional characterization of cytotoxic T lymphocytes in HIV-1 infected South African adultsPillay, Santhoshan Thiagaraj 12 1900 (has links)
Bibliography / Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In just 25 years since the first reported cases in 1981, the number of Human
Immunodeficiency virus (HIV) infected people has risen to 65 million, and over 25
million have died of acquired immunodeficiency syndrome (AIDS). Sub-Saharan Africa
accounts for 67% of all people living with HIV and 72% of deaths in this region were
AIDS related. Tuberculosis (TB) is one of the most common opportunistic infections in
AIDS patients, particularly in developing countries, where 60 - 70% of TB cases occur
in HIV-1-infected persons. HIV-1 is a high risk factor for the development of TB, the
reactivation of a latent Mycobacterium tuberculosis infection and also progressive TB.
CD8+ Cytotoxic T Lymphocytes (CTL) are pivotal in the host immune response to HIV
infection. CTL are associated with resolution of acute infection and with reduction in
viral load. Studies in macaques and humans indicate the importance of CTL in the
control of HIV infection, where reduction in CD8+ T cell number has been correlated
with progression to AIDS.
The current study was a cross-sectional descriptive study of CD8+ T cells of HIV+ adult
South Africans with and without TB co-infection (TB disease). The cohort consisted of
anti-retroviral therapy (ART) naive patients and all CTL analyses were carried out on
peripheral blood mononuclear cells (PBMCs). A total of 60 South African adults from the
Western Cape were utilized in this study, including 15 healthy controls; 30 HIV+TB-individuals
and 15 HIV+TB+ individuals. Expression of phenotypic, activation and
functional markers were investigated by flow cytometry with the use of fluorochomeconjugated
antibodies. The markers examined included the novel activation marker
CD137, the CTL associated markers Perforin, Granzyme A, CD107a/b, Fas (CD95),
and FasL (CD95L), intracellular cytokines IFN-y and TNF-a and the chronic HIV CTL
dysfunction marker PD-1. HIV infection alone was associated with increased baseline expression of TNF-a,
Perforin, Granzyme A, PD-1, Fas (CD95), and FasL (CD95L), but not CD137(4-1BB) or
IFN-y as compared to uninfected controls. TB co-infection resulted in further increased
baseline expression of TNF-a, perforin, PD-1, FasL (CD95L), as well as increased IFN-y. HIV-1 antigen (gag)-specific stimulation in vitro indicated that in HIV infection was
associated with antigen-specific upregulation of activation and cytotoxicity markers
CD137, IFN-y, TNF-a, Fas, FasL and CD107a/b. In TB co-infection a reduction in
antigen-specific degranulation (CD107a/b up-regulation) and also Fas and FasL
expression was observed.
TB co-infection (in the form of active pulmonary TB) reduced antigen-specific CTL
functional activity, but simultaneously there was an association with increased baseline
PD-1 expression and also cytolytic marker expression (Fas, FasL, TNF-a). These
cytolytic markers could be involved in non-antigen-specific bystander target cell death.
The expression of the co-stimulatory molecule CD137 appeared to correlate with
interferon-y production and levels of degranulation, confirming its usefulness as a
putative surrogate marker of functional responsiveness. These data indicate that in
addition to impacting on CD4 T cell function, TB co-infection leads to higher baseline
expression of CTL-associated markers, but to dysfunctional antigen-specific CTL
responses. / AFRIKAANSE OPSOMMING: Slegs vyf en twintig jaar na die eerste berigte van die menslike immuniteitsgebrekvirus
(MIV) in 1981, het die getal MIV-geinfekteerde individue gestyg tot 65 miljoen en het
meer as 25 miljoen mense alreeds gesterf aan die verworwe immuniteitsgebrek
sindroom (VIGS). Sub Sahara Afrika maak 67% uit van alle HIV gevalle en het `n MIVverwante
doodsyfer van 72%. Een van die algemeenste opportunistiese infeksies in
VIGS pasiente is Tuberkulose (TB). In ontwikkelende lande, veral, kom 60-70% van TB
gevalle voor in MIV-1 geinfekteerde individue. MIV-1 is `n hoe risiko faktor vir die
ontwikkeling van TB, die heraktivering van latente Mycobacterium tuberculosis infeksie
en progressiewe TB.
Die CD8+ sitotoksiese T Limfosiete (STL) se immuun reaksie teen `n MIV infeksie is
noodsaaklik en word geassosieer met `n resolusie van die akute infeksie en `n afname
in viruslading. Studies in die mens en macaque het getoon dat sitotoksiese T limfosiete
belangrik is vir die beheer van MIV infeksies aangesien die afname in CD8+ sel getalle
korreleer met die verloop tot VIGS.
Hierdie deursnit-beskrywende studie het die CD8+ T selle van MIV+ volwasse Suid-Afrikaners, met of sonder`n TB mede-infeksie, ondersoek. STL analise is gedoen op die
perifere bloed mono-nuklere selle (PBMS) van pasiente wat geen teen-retrovirale
terapie (TRT) ontvang het nie. `n Totaal van sestig Suid-Afrikaanse volwassenes van
die Wes-Kaap het deelgeneem aan die studie wat 15 gesonde kontroles; 30 MIV+TBen
15 MIV+TB+ individue ingesluit het. Die uitdrukking van fenotipiese, aktiverings en
funksionele merkers is ondersoek deur middel van vloeisitometrie en fluorochroomgekonjugeerde
teenliggaampies. Laasgenoemde het ingesluit die nuwe
aktiversingsmerker CD 137, die STL geassosieerde merkers Perforien en Gransiem A,
CD 107a/b, Fas (CD95) en FasL (CD95L), intrasellulere sitokiene IFN-y en TNF-a en
PD-1, die merker vir chroniese MIV CTL disfunksie. Daar is gevind dat `n TB mede-infeksie (in die vorm van aktiewe pulmonere TB) die
antigeen-spesifieke STL funksie verlaag en terselftertyd `n verhoging in die uitdrukking
van PD-1 en sitolitiese merkers (Fas, FasL, TNF-a) bewerkstellig. Hierdie sitolitiese
basislyn merkers is moontlik betrokke by die dood van nie-antigeen-spesifieke
omstander teiken selle. Die uitdrukking van die mede-stimulatoriese molekule CD 137
blyk om te korreleer met die produksie van STL IFN-y en die vlakke van degranulasie.
Dit bevestig die merker se bruikbaarheid as `n gewaande surrogaat merker vir
funksionele reaksies. Die data toon verder dat `n TB mede-infeksie nie net `n effek het
op die CD4 T sel funksie nie, dit lei ook tot `n verhoogde basislyn uitdrukking van STLgeassosieerde
merkers, maar met disfunksionele antigeen-spesifieke STL reaksies.
Hierdie studie het bepaal dat `n MIV infeksie verbind word met `n toename in die
basislyn uitdrukking van TNF-a, Perforien, Gransiem A, PD-1, Fas (CD95) en FasL
(CD95L). Dit is egter nie die geval wanneer die uitdrukking van CD 137 (4-1BB) of IFN-y
vergelyk word met nie-geinfekteerde kontroles. `n TB mede-infeksie het `n verdere
toename in die uitdrukking van TNF-a, Perforien, PD-1, FasL (CD95L) getoon, asook `n
verhoging in IFN-y vanaf die basislyn. In vitro MIV-1 antigeen (gag)-spesifieke
stimulasies het aangedui dat `n MIV infeksie met die antigeen-spesifieke op-regulasie
van aktiverings en sitotoksiese merkers CD137, IFN-y, TNF-a, Fas, FasL en CD107a/b
geassosieer word. In `n TB mede-infeksie, is `n verlaging van antigeen-spesifieke
degranulasie (CD 107a/b op-regulasie) asook die uitdrukking van Fas en FasL
waargeneem. / The Poliomyelitis Research Foundation / The National Health Laboratory Service
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Contribution de la forme nucléaire de l'uracile DNA glycosylase aux étapes précoces du cycle de réplication du virus de l'immunodéficience humaine de type 1 / Contribution of the nuclear form of the uracil DNA glycosylase during early steps of HIV-1 replication cycleHérate, Cécile 06 July 2015 (has links)
La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.
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Contribution de la forme nucléaire de l'uracile DNA glycosylase aux étapes précoces du cycle de réplication du virus de l'immunodéficience humaine de type 1 / Contribution of the nuclear form of the uracil DNA glycosylase during early steps of HIV-1 replication cycleHérate, Cécile 06 July 2015 (has links)
La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.
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Contribution de la forme nucléaire de l'uracile DNA glycosylase aux étapes précoces du cycle de réplication du virus de l'immunodéficience humaine de type 1 / Contribution of the nuclear form of the uracil DNA glycosylase during early steps of HIV-1 replication cycleHérate, Cécile 06 July 2015 (has links)
La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.
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