HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms

Abstract

In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5′ common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.

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

“…Consequently, the deletions of SL3 included flanking regions and, in our opinion, it is likely to be that many of the RNA mutants used in these studies adopted aberrant structures. More recent works using exact deletions of SL3 show more modest effects on packaging 6,31 , in agreement with our results. Furthermore, our data are totally consistent with the observation that deletions of SL1 have profound effect on gRNA packaging, while substitutions of the SL1 apical loop that affect gRNA dimerization have modest effects 30,33 .…”

“…2). 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 .…”

“…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 .…”

“…The trans-activation response element (TAR) stem-loop (SL) is essential for Tat-mediated activation of transcription; the poly(A) hairpin contains the 5 0 -copy of the polyadenylation signal and may also be involved in a long range pseudo-knot 26 ; the PBS domain is crucial for initiation of reverse transcription (RT); SL1 initiates dimerization of the gRNA thanks to the 6-nt self-complementary motif in its loop 2,3,27 ; SL2 contains the major splice donor site; SL3 is involved in RNA packaging (see below); the unstable SL4 involves the initiation codon of gag and may adopt alternative conformations. The region encompassing SL1 to SL4, usually referred to as 'Psi', and especially SL1 and SL3, are required for efficient packaging 6,11,[28][29][30][31][32][33] . Other studies showed that TAR 34 , the Poly(A) hairpin 35,36 and the PBS domain 37 are also required for optimal packaging of the HIV-1 gRNA.…”

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

“…It has been reported that the RNA has a crucial role in the structural integrity of the retroviral particle and that selective and non-selective packaging of cellular RNAs can occur both in murine leukemia virus and in HIV-1 particles. 41,44,45 Moreover, it has been shown that HIV-1-based vectors with deletions of SL1-SL4, the region encompassing sequences involved in targeting viral RNA into virions as high-affinity binding site for Gag, 46,47 can still be packaged and transduced, although with no apparent normal dimerization, suggesting the presence of a c-independent mechanism of packaging. 48 Thus, alternative and not mutually exclusive explanations have to be taken into account to explain the lack of high efficiency of pseudotyped HIV-1 particles transducing ability.…”