RESUMO
Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.
Assuntos
Apolipoproteínas E/fisiologia , Infecções por HIV/metabolismo , Macrófagos/metabolismo , Adulto , Apolipoproteínas/metabolismo , Apolipoproteínas E/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Humanos , Macrófagos/virologia , Masculino , Regulação para Cima , Replicação Viral/genética , Replicação Viral/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
RNA helicase plays an important role in host mRNA and viral mRNA transcription, transport, and translation. Many viruses utilize RNA helicases in their life cycle, while human immunodeficiency virus type 1 (HIV-1) does not encode an RNA helicase. Thus, host RNA helicase has been involved in HIV-1 replication. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether distinct DDX RNA helicases cross-talk and cooperate to modulate the HIV-1 Rev function. In this study, we noticed that distinct DDX RNA helicases, including DDX1, DDX3, DDX5, DDX17, DDX21, DDX56, except DDX6, bound to the Rev protein and they colocalized with Rev in nucleolus or nucleus. In this context, these DEAD-box RNA helicases except DDX6 markedly enhanced the HIV-1 Rev-dependent RNA export. Furthermore, DDX3 interacted with DDX5 and synergistically enhanced the Rev function. As well, combination of other distinct DDX RNA helicases cooperated to stimulate the Rev function. Altogether, these results suggest that distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function.
Assuntos
RNA Helicases DEAD-box/metabolismo , HIV-1/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Western Blotting , Nucléolo Celular/metabolismo , Nucléolo Celular/virologia , RNA Helicases DEAD-box/genética , Células HEK293 , HIV-1/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Microscopia Confocal , Ligação Proteica , Transporte de RNA , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function.
Assuntos
RNA Helicases DEAD-box/metabolismo , HIV-1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Citoplasma/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Long interspersed element-1 (LINE-1, L1) is a mobile genetic element comprising about 17% of the human genome. L1 utilizes an endonuclease to insert L1 cDNA into the target genomic DNA, which induces double-strand DNA breaks in the human genome and activates the DNA damage signaling pathway, resulting in the recruitment of DNA-repair proteins. This may facilitate or protect L1 integration into the human genome. Therefore, the host DNA repair machinery has pivotal roles in L1 mobility. In this study, we have, for the first time, demonstrated that the DNA repair protein, Rad18, restricts L1 mobility. Notably, overexpression of Rad18 strongly suppressed L1 retrotransposition as well as L1-mediated Alu retrotransposition. In contrast, L1 retrotransposition was enhanced in Rad18-deficient or knockdown cells. Furthermore, the Rad6 (E2 ubiquitin-conjugated enzyme)-binding domain, but not the Polη-binding domain, was required for the inhibition of L1 retrotransposition, suggesting that the E3 ubiquitin ligase activity of Rad18 is important in regulating L1 mobility. Accordingly, wild-type, but not the mutant Rad18-lacking Rad6-binding domain, bound with L1 ORF1p and sequestered with L1 ORF1p into the Rad18-nuclear foci. Altogether, Rad18 restricts L1 and Alu retrotransposition as a guardian of the human genome against endogenous retroelements.