An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

Abstract

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5′ splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

HIV-1 Pr55Gagbinds genomic and spliced RNAs with different affinity and stoichiometry

“…This result together with previous findings clearly points out how long-range interactions are necessary for gRNA specific encapsidation. 20,21,23,24,32,[39][40][41][42]69,79,107,108 Even though further analysis will be necessary to identify the nucleotides that are involved, our model indicates that this interaction is not possible in svRNAs (which differ from gRNA downstream of SL2, Fig. 2), and this could impact on Pr55 Gag discrimination between unspliced gRNA and svRNAs.…”

“…1A). Those elements were also found to impact gRNA packaging, [39][40][41][42] although one should be cautious in the interpretation of these results since mutations in the leader RNA could induce misfolding, thus indirectly affecting the dimerization and the packaging. 43,44 Interestingly, svRNAs also contain the RNA motifs located upstream of SL2 45 that are known to act as positive packaging signals in the gRNA such as SL1.…”

Mutations generated in human immunodeficiency virus type 1 long terminal repeat during vertical transmission correlate with viral gene expression

“…We found that 10 clones (MB9, IB2, IB5, IB9, IG9, IG16, MH3, MH12, IH2-3, IH2-7) had mutations in the TAR region with almost half of them showing mutations within the loop region and two clones showing mutations downstream of TAR. It has been reported that mutations in the hexanucleotide loop region of TAR decreases the level of cooperative Cyclin T1 and Tat binding (Helga-Maria et al, 1999). In addition, mutations in TAR region may affect secondary stem loop structure (Das et al, 1997;Feng and Holland, 1988) and decrease Tat and cellular factor binding to the TAR.…”

“…Within U3 there are three functional regions, the promoter (positions -78 to -1) which contains the TATAA box, the enhancer (-105 to -79) which is comprised of two NFκB transcription factor binding sites and three GC rich SpI sites (Gaynor, 1992), and the modulatory region (-454 to -104) which contains two AP-I sites, two NFAT sites, one NF-IL6 site, one USF-1 site, and one Ets-1 site. The R (+1 to +99) region contains a unique enhancer element termed TAR (+1 to +60), or transactivation response element, which is important for Tat activation, efficient reverse transcription, and efficient packaging of viral genomic RNA (Feng and Holland, 1988;Harrich et al, 2000;Helga-Maria et al, 1999). While LTR diversity in infected individuals has been examined (Blackard et al, 2000;Blackard et al, 2001), there has been no systematic study performed that correlates mutations generated during vertical transmission with HIV-1 gene expression.…”

Interaction-mediating sequences within class I viral fusion glycoproteins : their roles in viral infection and in applications

“…The minimal region required for autonomous HIV-1 RNA packaging during viral assembly is mapped to a region covering the first 350-400 nt of the genome, spanning the LTR and the 5' part of gag gene [258][259][260][261][262]. This means that even if pNL1.5EU+ contains the LTR promoter and the viral packaging (ψ) signal, its unspliced Envencoding transcript under the LTR promoter is unlikely to be packaged into the budding virions effectively.…”