RESUMO
We previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the human T cell leukemia virus 1 (HTLV-1) Tax protein, a known activator of cellular NF-κB. The rescue of integrase (IN)-deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-κB to the two NF-κB binding sites found in the HIV-1 LTR enhancer. Here, we report that the recruitment of NF-κB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN- HIV-1 mutant lacking both LTR NF-κB sites is entirely nonresponsive to Tax and fails to undergo the epigenetic changes listed above. Interestingly, we found that induction of Tax expression at 24 h postinfection, when unintegrated HIV-1 DNA is already fully repressed by inhibitory chromatin modifications, is able to effectively reverse the epigenetic silencing of that DNA and rescue viral gene expression. Finally, we report that heterologous promoters introduced into IN-deficient HIV-1-based vectors are transcriptionally active even in the absence of Tax and do not increase their activity when the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, as has been recently proposed. IMPORTANCE Integrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN- lentiviral vectors are able to achieve only low levels of gene expression, and methods to increase this low level have not been extensively explored. Here, we analyzed how the HTLV-1 Tax protein is able to rescue the replication of IN- HIV-1 in T cells, and we describe IN- lentiviral vectors, lacking any inserted origin of replication, that are able to express a heterologous gene effectively.
Assuntos
Regulação Viral da Expressão Gênica/genética , Produtos do Gene tax/metabolismo , Integrase de HIV/genética , HIV-1/genética , Integração Viral/genética , Linhagem Celular , DNA Viral/genética , Ativação Enzimática/genética , Expressão Gênica/genética , Células HEK293 , Integrase de HIV/deficiência , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Provírus/genética , Replicação Viral/genéticaRESUMO
Integration of HIV-1 linear DNA into the host chromatin is an essential step in the viral life cycle. However, the majority of reverse-transcribed, nuclear-imported viral genomes remain episomal, either as linear or circular DNA. To date, these nonintegrated viral genomes are largely considered "dead-end products" of reverse transcription. Indeed, limited gene expression from nonintegrated HIV-1 has been reported, although the mechanism that renders nonintegrating HIV-1 genomes incapable of supporting efficient viral replication has not been fully elucidated. Here, we demonstrate that nonintegrating HIV-1 and HIV-1-based vector genomes are organized into chromatin structures and enriched with histone modifications typical of transcriptionally silenced chromatin. Gene expression and replication of nonintegrating HIV-1 was notably increased in vitro upon exposure to histone deacetylase inhibitors (HDACi) in the form of various short-chain fatty acids (SCFAs) known to be endogenously produced by normal microbial-gut flora. Furthermore, we demonstrated genetic and functional crosstalk between episomal and integrated vector/viral genomes, resulting in recombination between integrated and nonintegrated HIV-1, as well as mobilization of episomal vector genomes by productive viral particles encoded by integrated viral genomes. Finally, we propose a mechanism describing the role of episomal HIV-1 forms in the viral life cycle in a SCFA-rich gut environment.
Assuntos
Epigênese Genética/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Trato Gastrointestinal/microbiologia , Genoma Viral/genética , Infecções por HIV/virologia , HIV-1/genética , Integração Viral/efeitos dos fármacos , Cromatina/metabolismo , Inativação Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Genoma Viral/efeitos dos fármacos , Infecções por HIV/genética , Integrase de HIV/deficiência , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Monócitos/citologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV G protein proved inefficient even in mitotically active primary B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24% +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave different results. Freshly isolated, nondividing myeloma cells were efficiently transduced by HIV vectors; for 6 myelomas the range was 14% to 77% (median, 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in primary B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in primary human B cells and myeloma cells for the purposes of research and the development of gene therapies.