RESUMO
Current HIV-1 gene therapy approaches aim at stopping the viral life cycle at its earliest steps, such as entry or immediate postentry events. Among the most widely adopted strategies are CCR5 downregulation/knockout and the use of broadly neutralizing antibodies. However, the long-term efficacy and side effects are still unclear. TRIM5α is an interferon-stimulated restriction factor that can intercept incoming retroviruses within one hour of cytosolic entry and potently inhibit the infectivity of restriction-sensitive viruses. The human TRIM5α (TRIM5αhu) generally does not efficiently target HIV-1, but point mutations in its capsid-binding domain can confer anti-HIV-1 activity. Although the mechanisms by which TRIM5αhu mutants inhibit HIV-1 are relatively well understood, their characterization as potential transgenes for gene therapy is lacking. Additionally, previous reports of general immune activation by overexpression of TRIM5α have hindered its broad adoption as a potential transgene. Here we demonstrate the ability of the R332G-R335G TRIM5αhu mutant to efficiently restrict highly divergent HIV-1 strains, including Group O, as well as clinical isolates bearing cytotoxic T lymphocyte escape mutations. R332G-R335G TRIM5αhu efficiently protected human lymphocytes against HIV-1 infection, even when expressed at relatively low levels following lentiviral transduction. Most importantly, under these conditions Rhesus macaque TRIM5α (TRIM5αRh) and TRIM5αhu (wild-type or mutated) had no major effects on the NF-κB pathway. Transgenic TRIM5α did not modulate the kinetics of IκBα, JunB, and TNFAIP3 expression following TNF-α treatment. Finally, we show that human lymphocytes expressing R332G-R335G TRIM5αhu have clear survival advantages over unmodified parental cells in the presence of pathogenic, replication-competent HIV-1. These results support the relevance of R332G-R335G and other mutants of TRIM5αhu as candidate effectors for HIV-1 gene therapy.
Assuntos
Proteínas de Transporte/genética , Terapia Genética , Infecções por HIV/genética , HIV-1/genética , Proteínas Mutantes/genética , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/uso terapêutico , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Lentivirus/genética , Linfócitos/patologia , Linfócitos/virologia , Macaca mulatta , Proteínas Mutantes/uso terapêutico , Mutação , Ligação Proteica , Transgenes , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathies (STM) and in Bardet-Biedl syndrome. Few cases have been described to date in LGMD2H/STM, but this gene is not systematically analysed because of the absence of specific signs and difficulties in protein analysis. By using high-throughput variants screening techniques, we identified variants in TRIM32 in two patients presenting nonspecific LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 because association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that TRIM32/ASTN2 inactivation could be associated with cognitive impairment.
Assuntos
Hibridização Genômica Comparativa/métodos , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Distrofia Muscular do Cíngulo dos Membros/genética , Fatores de Transcrição/genética , Adulto , Sequência de Bases , Análise Mutacional de DNA/métodos , Saúde da Família , Feminino , Humanos , Masculino , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-ß, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-ß mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection.