RESUMO
Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.
Assuntos
Fator de Iniciação 4A em Eucariotos/genética , HIV-1/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , RNA Mensageiro/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Linhagem Celular , Fator de Iniciação 4A em Eucariotos/metabolismo , HIV-1/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Ligação Proteica , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention.
Assuntos
RNA Helicases DEAD-box/genética , Infecções por HIV/genética , HIV-1/genética , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/genética , Transporte Ativo do Núcleo Celular/genética , Regulação Viral da Expressão Gênica , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/genética , Proteína Exportina 1RESUMO
Post-transcriptional control in both HIV-1 and HIV-2 is a highly regulated process that commences in the nucleus of the host infected cell and finishes by the expression of viral proteins in the cytoplasm. Expression of the unspliced genomic RNA is particularly controlled at the level of RNA splicing, export, and translation. It appears increasingly obvious that all these steps are interconnected and they result in the building of a viral ribonucleoprotein complex (RNP) that must be efficiently translated in the cytosolic compartment. This review summarizes our knowledge about the genesis, localization, and expression of this viral RNP.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/fisiologia , HIV-2/fisiologia , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , RNA Viral/metabolismo , Replicação Viral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , HIV-1/genética , HIV-2/genética , Humanos , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismoRESUMO
During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells. This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding protein and stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observed that increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein.
Assuntos
Grânulos Citoplasmáticos/virologia , HIV-2/genética , RNA Viral/metabolismo , Montagem de Vírus , Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Viral , HIV-2/fisiologia , Células HeLa , Humanos , Biossíntese de Proteínas , RNA Viral/análise , RNA Viral/biossíntese , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
DDX3 belongs to the DEAD-box proteins, a large family of ATP-dependent RNA helicases that participate in all aspects of RNA metabolism. Human DDX3 is a component of several messenger ribonucleoproteins that are found in the spliceosome, the export and the translation initiation machineries but also in different cytoplasmic mRNA granules. DDX3 has been involved in several cellular processes such as cell cycle progression, apoptosis, cancer, innate immune response, and also as a host factor for viral replication. Interestingly, not all these functions require the catalytic activities of DDX3 and thus, the precise roles of this apparently multifaceted protein remain largely obscure. The aim of this review is to provide a rapid and critical overview of the structure and functions of DDX3 with a particular emphasis on its role during mRNA metabolism.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , RNA Helicases DEAD-box/química , HumanosRESUMO
The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.
Assuntos
Infecções por Hantavirus/virologia , Orthohantavírus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Linhagem Celular , Regulação Viral da Expressão Gênica , Orthohantavírus/metabolismo , Humanos , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
In the process of translation of eukaryotic mRNAs, the 5' cap and the 3' poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3' poly(A) tail. Here we report that the 3' untranslated region (3'UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3'UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein.