Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

Abstract

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

“…Because the ED-deficient HIV-1 mutants J8 and J10 package normal genomic RNA levels, the results suggest that the early KL dimer form is sufficient to trigger efficient genome packaging, which is consistent with previous reports (23,24,75,76). The inability of these mutant HIV-1 RNA genomes to mature into more stable ED dimers may trigger subsequent defects, e.g.…”

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

“…The 410-fold reduced binding of Pr55 Gag to spliced RNAs compares well with quantitative packaging studies showing that gRNA is incorporated 50-100-fold more efficiently than spliced RNAs into HIV-1 viral particles 6 . Thus, selection during the initial binding event appears to be the main factor governing selective packaging of the gRNA, even though other pathways such as the gRNA nuclear export pathway and subcellular localization might also play a significant role 7,8 . Our RNA binding data is also consistent with the many viral replication studies showing that although the NC domain is crucial for the selectivity of the packaging process, other domains of Pr55 Gag are also involved [18][19][20][21][22][23] .…”

“…HIV-1 gRNA and spliced vRNAs compete for a common packaging pathway, while cellular RNAs are usually packaged with low efficiency, through a different mechanism 6 . It is currently unclear whether discrimination between gRNA and spliced RNA is mediated by the initial binding step to Pr55 Gag , or whether other pathways such as the gRNA nuclear export pathway and subcellular localization are involved 7,8 .…”

Cell biology of retroviral RNA packaging

“…By coupling protein cross-linking experiments with membrane flotation approaches, it was shown that Gag forms recognized by MS2 fused to GFP. Detection of MS2-GFP-bound RNA in VLPs collected from cell culture supernatant, either by microscopy 61,70,71 or by protein gel blotting analysis, 68 indicated that the modified genomes were efficiently packaged into virions. Importantly, this encapsidation was dependent on the RNA packaging signal.…”

“…[89][90][91][92] For HIV-1 RNA, dimerization is likely to happen later in the cytoplasm. 71 However, where exactly in the cytoplasm HIV-1 dimerization occurs and when it occurs relative to Gag binding remained to be elucidated.…”