RESUMO
In mammals, 2'-O-methylation of RNA is a molecular signature by which the cellular innate immune system distinguishes endogenous from exogenous messenger RNA1-3. However, the molecular functions of RNA 2'-O-methylation are not well understood. Here we have purified TAR RNA-binding protein (TRBP) and its interacting partners and identified a DICER-independent TRBP complex containing FTSJ3, a putative 2'-O-methyltransferase (2'O-MTase). In vitro and ex vivo experiments show that FTSJ3 is a 2'O-MTase that is recruited to HIV RNA through TRBP. Using RiboMethSeq analysis4, we identified predominantly FTSJ3-dependent 2'-O-methylations at specific residues on the viral genome. HIV-1 viruses produced in FTSJ3 knockdown cells show reduced 2'-O-methylation and trigger expression of type 1 interferons (IFNs) in human dendritic cells through the RNA sensor MDA5. This induction of IFN-α and IFN-ß leads to a reduction in HIV expression. We have identified an unexpected mechanism used by HIV-1 to evade innate immune recognition: the recruitment of the TRBP-FTSJ3 complex to viral RNA and its 2'-O-methylation.
Assuntos
HIV-1/imunologia , HIV-1/patogenicidade , Imunidade Inata , Metiltransferases/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , HIV-1/genética , Células HeLa , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/deficiência , Ligação Proteica , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismoRESUMO
The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.
Assuntos
DNA/imunologia , Herpesvirus Humano 8/imunologia , Imunidade Inata , RNA Longo não Codificante/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/imunologia , Autoantígeno Ku/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/imunologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/imunologia , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/imunologia , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição , TransfecçãoRESUMO
Small non-coding RNAs of 18-25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3' end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.
Assuntos
HIV-1/genética , RNA de Cadeia Dupla/metabolismo , RNA não Traduzido/química , RNA Viral/metabolismo , Linhagem Celular , HIV-1/fisiologia , Humanos , RNA de Transferência de Lisina/metabolismo , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Linfócitos T/virologia , Replicação ViralRESUMO
Hidradenitis suppurativa (HS) is a chronic, relapsing, inflammatory skin disease. HS appears to be a primary abnormality in the pilosebaceous-apocrine unit. In this work, we characterized hair follicle stem cells (HFSCs) isolated from HS patients and more precisely the outer root sheath cells (ORSCs). We showed that hair follicle cells from HS patients had an increased number of proliferating progenitor cells and lost quiescent stem cells. Remarkably, we also showed that the progression of replication forks was altered in ORSCs from hair follicles of HS patients, leading to activation of the ATR/CHK1 pathway. These alterations were associated with an increased number of micronuclei and with the presence of cytoplasmic ssDNA, leading to the activation of the IFI16/STING pathway and the production of type I IFNs. This mechanistic analysis of the etiology of HS in the HFSC compartment establishes a formal link between genetic predisposition and skin inflammation observed in HS.
Assuntos
Dano ao DNA , Replicação do DNA , Folículo Piloso/metabolismo , Hidradenite Supurativa/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco/metabolismo , Adolescente , Adulto , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Feminino , Folículo Piloso/patologia , Hidradenite Supurativa/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco/patologiaRESUMO
The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/fisiologia , MicroRNAs/metabolismo , RNA Viral/metabolismo , Replicação Viral/fisiologia , Proteínas Argonautas , Linhagem Celular , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Polirribossomos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Latência Viral/fisiologiaRESUMO
The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.
Assuntos
HIV-1 , Interleucina-10/biossíntese , Monócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Acetilação , Núcleo Celular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Citoplasma/metabolismo , Histonas/metabolismo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Quinase Induzida por NF-kappaBRESUMO
HIV-1 Tat protein stimulates the production of both TNF-alpha and IL-10 in human monocytes. Taking into account the ability of TNF-alpha to induce IL-10 production, we evaluated the link between Tat, TNF-alpha and IL-10 and the implication of PKC and p38 MAP kinase pathways. Our data showed that (i) in the presence of neutralizing anti-TNF-alpha antibodies, IL-10 production is only partially inhibited; (ii) in a calcium-free medium, while TNF-alpha production is totally inhibited, Tat continues to induce IL-10; (iii) under these conditions, Tat-mediated IL-10 production is associated with PKC-delta activation; and (iv) downstream of PKC, p38 MAP kinase is crucial for TNF-alpha independent IL10 production. Overall, our data suggest a new mechanism, implicating Tat protein, by which HIV-1 may maintain a constant production of the immunosuppressive IL-10 cytokine, even in the absence of TNF-alpha production. In consequence, HIV-1 may escape immune surveillance and thus promote the establishment of an immunosuppressive state.
Assuntos
Interleucina-10/imunologia , Monócitos/imunologia , Proteína Quinase C-delta/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Cálcio/metabolismo , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Monócitos/citologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-delta/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Frações Subcelulares/enzimologia , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
MicroRNAs (miRNAs) are non-coding small RNAs that play important roles in a variety of biological pathways including cellular proliferation and apoptosis. Recent studies have linked the expression of selected miRNAs to carcinogenesis and viral pathogenesis. Here, we will discuss examples of roles served by cellular miRNAs and virus-encoded miRNAs in the development of cancers and viral diseases.
Assuntos
MicroRNAs/fisiologia , Neoplasias/etiologia , Viroses/etiologia , Apoptose , Proliferação de Células , Humanos , Neoplasias/genética , Viroses/genéticaRESUMO
Inside eukaryotic cells, small RNA duplexes, called small interfering RNAs (siRNAs), activate a conserved RNA interference (RNAi) pathway which leads to specific degradation of complementary target mRNAs through base-pairing recognition. As with other viruses, studies have shown that replication of the HIV-1 in cultured cells can be targeted and inhibited by synthetic siRNAs. The relative ease of siRNA design and the versatility of RNAi to target a broad spectrum of mRNAs have led to the promise that drug discovery in the RNAi pathway could be effective against pathogens. This review discusses the current experimental principles that guide the application of RNAi against HIV and describes challenges and limitations that need to be surmounted in order for siRNAs to become practical antiviral drugs. The practical use of RNAi therapy for HIV infection will depend on overcoming several challenges, including the ability to establish long-term expression of siRNA without off-target effects and the capacity to counteract mutant escape viruses.
Assuntos
Infecções por HIV/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Humanos , Interferência de RNA , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain.
Assuntos
Produtos do Gene tat/metabolismo , HIV-1/metabolismo , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Proteínas Argonautas , Fator de Iniciação 2 em Eucariotos , Produtos do Gene tat/genética , HIV-1/enzimologia , HIV-1/genética , Humanos , MicroRNAs/genética , Fatores de Iniciação de Peptídeos/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Viral/genética , Complexo de Inativação Induzido por RNA/genética , Ribonuclease III/genética , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Initially reported for Caenorhabditis elegans, microRNA (miRNA) has been shown to regulate gene expression in plants, flies, and mammals . Here, we report on our approaches to investigate the role of miRNAs in human immunodeficiency virus (HIV)-1 infection. Using computer-directed foldings, we first identify potential sequences in HIV-1 that putatively encode miRNAs. Subsequently, we use Northern blotting of RNAs isolated from HIV-infected cells to confirm expression of predicted miRNA sequences. Finally, we use a scanning algorithm to search 3' untranslated regions (UTRs) of human messenger RNAs (mRNAs) in the attempt to predict potential sites targeted by HIV-1 miRNAs.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , MicroRNAs/genética , Algoritmos , Biologia Computacional/métodos , Humanos , MicroRNAs/química , MicroRNAs/metabolismo , Análise em Microsséries/métodosRESUMO
MicroRNAs (miRNAs) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating a variety of biological processes. Recent studies suggest that cellular miRNAs may serve to control the replication of viruses in cells. If such is the case, viruses might be expected to evolve the ability to modulate the expression of cellular miRNAs. To ask if expression of HIV-1 genes changes the miRNA profiles in human cells, we employed a high throughput microarray method, termed the RNA-primed Array-based Klenow Enzyme (RAKE) assay. Here, we describe the optimization of this assay to quantify the expression of miRNAs in HIV-1 transfected human cells. We report distinct differences in miRNA profiles in mock-transfected HeLa cells versus HeLa cells transfected with an infectious HIV-1 molecular clone, pNL4-3.
Assuntos
Perfilação da Expressão Gênica , HIV-1/genética , MicroRNAs/análise , Células HeLa , Humanos , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).
Assuntos
Infecções por HIV/metabolismo , HIV/fisiologia , MicroRNAs/fisiologia , RNA Interferente Pequeno/fisiologia , RNA Viral/fisiologia , HIV/genética , Infecções por HIV/genética , Humanos , MicroRNAs/isolamento & purificação , RNA Interferente Pequeno/isolamento & purificação , RNA Viral/isolamento & purificaçãoRESUMO
In HIV-infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with the disease progression toward AIDS. We have previously shown that HIV-1 Tat induces IL-10 production by human monocytes via a protein kinase C (PKC) -dependent pathway. Here we show that PKC activation by Tat is essential for IL-10 induction. Among the eight PKC isoforms present in human monocytes, we investigated which isoform(s) plays this crucial role in Tat-mediated IL-10 production and show that 1) Tat can activate PKC-alpha, PKC-betaII, PKC-delta, and PKC-epsilon, 2) of these four potential candidates, only PKC-betaII, PKC-delta, and PKC-epsilon are activated by the active domain Tat 1-45, which is responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-alpha and PKC-epsilon by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-betaII or PKC-delta partially inhibits IL-10 production; and 4) the simultaneous inhibition of PKC-betaII and PKC-delta totally inhibits Tat-mediated IL-10. Altogether, these results suggest that the induction of IL-10 by Tat is strictly dependent on the PKC-delta and -betaII isoforms.
Assuntos
Produtos do Gene tat/farmacologia , HIV-1 , Interleucina-10/biossíntese , Monócitos/imunologia , Proteína Quinase C/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Monócitos/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Proteína Quinase C-delta , Transporte Proteico , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.
Assuntos
Produtos do Gene nef/imunologia , Anticorpos Anti-HIV/biossíntese , Antígenos HIV/imunologia , HIV-1/imunologia , Ácido Láctico/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Polímeros/administração & dosagem , Animais , Células Cultivadas , Citocinas/biossíntese , Epitopos de Linfócito B/imunologia , Feminino , Adjuvante de Freund/farmacologia , Produtos do Gene nef/administração & dosagem , Antígenos HIV/administração & dosagem , Imunização , Imunoglobulina G/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Baço/imunologia , Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
MicroRNAs (miRNAs) are small RNAs of 21-25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit.
Assuntos
HIV-1/genética , MicroRNAs/genética , RNA Viral/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Genoma Humano , Genoma Viral , Humanos , Processamento Pós-Transcricional do RNARESUMO
The effect of HIV-1 Tat protein on the production of IL-10, an immunosuppressive cytokine, was examined in human primary monocytes obtained from healthy HIV-1-negative blood donors. As expected and in agreement with our previous data, a dose-dependent induction of IL-10 was observed. In addition, we showed that this induction is mediated by the PKC pathway: in the presence of Ro 31-8220, an inhibitor of all PKC isozymes, or after 48 h of PMA treatment, Tat protein becomes unable to stimulate IL-10 production. Among the 11 PKC isozymes, eight (PKC alpha, beta(I), beta(II), delta, epsilon, eta, zeta, mu) are expressed in monocytes. In this study, by analyzing the translocation to the membrane after Tat stimulation, we showed that PKC alpha, beta(I), beta(II), delta and epsilon isozymes are activated by Tat. Moreover, by combining different approaches including selective PKC inhibitors (Gö6983, Gö6976, hispidin and rottlerin), we showed that PKC beta(II) and delta isozymes are essential for the activation of IL-10 production in human monocytes following stimulation by HIV-1 Tat protein.
Assuntos
Produtos do Gene tat/metabolismo , HIV-1/patogenicidade , Interleucina-10/biossíntese , Monócitos/imunologia , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Carbazóis/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Indóis/farmacologia , Maleimidas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-delta , Pironas/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaAssuntos
HIV-1/genética , Interferência de RNA/fisiologia , Fármacos Anti-HIV/farmacologia , Infecções por HIV/genética , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , MicroRNAs/metabolismo , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA-exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.
Assuntos
RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Carioferinas/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribonuclease III/genética , Adenoviridae/genética , Adenoviridae/fisiologia , Infecções por Adenoviridae/virologia , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Carioferinas/genética , MicroRNAs/genética , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Viral/genética , Ribonuclease III/metabolismo , Replicação ViralRESUMO
A role for microRNAs (miRNA) in human T-cell leukemia virus 1 (HTLV-1)-mediated cellular transformation has not been described. Here, we profiled miRNA expression in HTLV-1-transformed human T-cell lines and primary peripheral blood mononuclear cells from adult T-cell leukemia patients. Analyses of 11 different profiles revealed six miRNAs that were consistently up-regulated. Two of the up-regulated miRNAs (miR-93 and miR-130b) target the 3' untranslated region (3'UTR) of the mRNA for a tumor suppressor protein, tumor protein 53-induced nuclear protein 1 (TP53INP1). A low expression level of TP53INP1 protein was found in HTLV-1-transformed cells. Additionally, when antagomirs were used to knock down miR-93 and miR-130b in these cells, the expression of TP53INP1 was increased, suggesting that the latter is regulated inside cells by the former. A role for TP53INP1 in regulating cell growth was established by experiments that showed that enhanced TP53INP1 expression increased apoptosis. Collectively, the findings implicate a miR-93/miR-130b-TP53INP1 axis that affects the proliferation and survival of HTLV-1-infected/transformed cells.