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

Abstract

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.

Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

“…[16]. We chose to leave the 5’ leader region intact because it modulates HIV-1 expression by specifying nucleosome and transcription factor binding [9], folds into a finely-balanced equilibrium of RNA elements that regulate RNA fates [41], and is highly sensitive to mutation [42]. B-HIVE vectors encode LTR-driven GFP but no virus structural proteins.…”

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

“…Deletion of nt 22 to 378 in gag does not substantially decrease infectious-virus production (15,47), indicating that there are no essential cis-acting elements in the region. However, altering the RNA sequence could modulate the local RNA structure in ways that a large deletion did not, and the 5= region of gag has been shown to indirectly regulate gRNA packaging by regulating the structure of the 5= UTR (48,49).…”

NMR Studies of Retroviral Genome Packaging

“…We note here that some HIV-1 NL4-3 vectors with non-native residues downstream of the leader were also poorly packaged. This was originally attributed to a role of coding residues in packaging; however, more recent studies indicate that the non-native residues might disrupt the structure of the leader and thereby inhibit packaging [ 42 ]. It is, thus, possible that the vectors employed in these early MoMuLV packaging experiments might also adversely affect the structure and function of the packaging signal.…”

“…Although it is unclear where in the cell NC–RNA interactions initially occur, confocal studies showed that virus assembly is nucleated by the binding of a small number of Gag proteins (possibly a dozen or fewer [ 29 ]) to the viral genome, at plasma membrane assembly sites [ 30 ], which leads to the subsequent recruitment of several thousand additional Gag proteins [ 29 , 31 ] and cellular factors that promote virus budding [ 32 , 33 ]. Although virions can assemble in the absence of their genomes by incorporating an equivalent amount of cellular RNAs [ 34 , 35 , 36 , 37 , 38 , 39 ], vector RNAs containing the authentic packaging signal can efficiently out-compete these more abundant RNAs for packaging [ 40 , 41 ], as long as the non-native downstream vector residues do not interfere with proper folding of the packaging signal [ 42 ].…”

“…Dimerization, packaging, and other RNA-dependent functions (including transcriptional activation, splicing, and initiation of reverse transcription) are promoted by the elements located within the 5′-leader of the RNA [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 16 , 17 ], which is among the most conserved regions of the genome [ 55 , 56 ], ( ). Although some studies with mutant genomes suggested that residues in the gag open reading frame might also be important for packaging, it now appears that packaging defects were caused by misfolding of the 5′-leader due to interactions with non-native downstream sequences [ 42 ]. Studies identified regions within the 5′-leaders of HIV-1 [ 40 ], MoMuLV [ 57 , 58 ], and Rous Sarcoma Virus (RSV) [ 59 , 60 , 61 , 62 , 63 , 64 ], which are independently capable of directing heterologous RNAs into assembling virus-like particles (VLPs).…”