RESUMEN
In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estrés Fisiológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosforilación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , Biosíntesis de Proteínas , Regulación Fúngica de la Expresión Génica , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Transducción de Señal , Ribosomas/metabolismo , Ribosomas/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genéticaRESUMEN
Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA +/+ ) and CypA-depleted (CypA -/- ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA +/+ cells but not in CypA -/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA +/+ cells but not in CypA -/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA -/- cells had lower activity in vitro compared to those from CypA +/+ cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA +/+ and CypA -/- cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. Importance: HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.