Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5′ leader Ψ elements plus poorly defined additional features. We previously defined minimal 5′ leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5′ leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5′ leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination.     

A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction

Replication of HIV-1 depends on the viral Tat protein, which functions via a target sequence, TAR, present in the proviral long terminal repeat (LTR) and at the 5' end of viral mRNAs. We have shown that Tat potentiates the expression of TAR-containing RNAs, but only when Tat and the TAR-containing RNA are present in the nucleus. We now show that a small change in the TAR loop abolishes nuclear potentiation by Tat. Furthermore, the HIV-1 U3 region induces expression incompetence in mRNA synthesized by this promoter. RNAs of identical structure are, however, translated efficiently when produced from the CMV-IE promoter. The Tat-TAR system appears, therefore, to rescue the expression potential of HIV-1 LTR-directed RNA.