RESUMEN
A genetic screen previously identified the N-terminal 91 amino acids of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication. Overexpression of N91-eIF3f or full-length eIF3f reduced the level of HIV-1 mRNAs in the infected cell. Here we show that N91-eIF3f and eIF3f act by specifically blocking the 3' end processing of the HIV-1 pre-mRNA both in vivo and in vitro. Furthermore, the results suggest that eIF3f mediates this restriction of HIV-1 expression through the previously unsuspected involvement of a set of factors that includes eIF3f, the SR protein 9G8, and the cyclin-dependent kinase 11 (CDK11). eIF3f affects HIV-1 3' end processing by modulating the sequence-specific recognition of the HIV-1 pre-mRNA by 9G8.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , VIH-1/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Procesamiento de Término de ARN 3'/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Extractos Celulares , Núcleo Celular/metabolismo , Factor 3 de Iniciación Eucariótica/química , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Nucleares , Poli A/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina , Replicación ViralRESUMEN
Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3' long terminal repeat (3'LTR) region in the viral mRNA. We show that the 3' end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3' end processing of HIV-1 mRNAs.