Abstract
The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.
Highlights
Human immunodeficiency virus type 1 (HIV-1) is unique in that it produces two RNA types: Rev-independent and Revdependent
The Renilla luciferase (Rluc) RNA levels increased to the levels of the psiCHECK control in the presence of Rev-HA (Fig. S2D, Vectors 1, 2, 9 and 10, red arrows). These results suggested that Rev transported these Rev response element (RRE)-containing mRNAs; the increase in the level of transported mRNAs did not exceed the levels observed in the psiCHECK control, which suggested that further upregulation of the observed Rluc activities (Vectors 1, 2, 9 and 10 in Fig. 1C, red arrows) was dependent on translational-level Rev function
Unspliced mRNA was required to augment expression and mediate miRNA silencing (Fig. 1E), which appears to be in contrast with the instability elements (INS)/cis-acting repressive sequences (CRS) effect
Summary
Human immunodeficiency virus type 1 (HIV-1) is unique in that it produces two RNA types: Rev-independent and Revdependent. Rev appears to enhance translation [2,3] and packaging efficiency [4,5,6] in the cytoplasm. The unspliced HIV-1 RNAs are translated as Gag and Gag-Pol polyproteins or are packaged as genomes into viral particles. Both Gag and the viral genome are transported in late endosomal structures called multivesicular bodies (MVBs) to the plasma membrane for virus egress, a process that requires the participation of the Endosomal Sorting Complex Required for Transport (ESCRT) proteins [7,8,9,10,11]. The HIV-1 genome must be sequestered from the cellular translational machinery to be packaged into viral particles
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