A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly

Abstract

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

A Survey of DDX21 Activity During Rev/RRE Complex Formation

“…In both cases, we observed similar bi-exponential kinetic behavior: rate constants 1.06 ± 0.20 s −1 and 0.16 ± 0.06 s −1 in the presence of DDX21 (Figure 8d left) and 1.38 ± 0.18 s −1 and 0.11 ± 0.02 s −1 in the presence of DDX1 (Figure 8d right). In both cases, the fast phase is predominant, whereas the slow phase corresponds to the Rev binding rate observed in the absence of any DDX protein (0.09 ± 0.01 s −1 ), agreeing with previous results [23]. These results indicate that DDX1 and DDX21 both accelerate binding of the first Rev monomer to the RRE and likely act by a similar mechanism.…”

“…As demonstrated previously, the presence of DDX1 increases the proportion of fluorescent signal at higher intensity values, indicating an increase in the number of Rev monomers associated with each RRE molecule (Figure 8c, bottom right panel)[46]. Recently, we showed that DDX1 acts through the RRE RNA to promote Rev-RRE assembly [23, 24]. This ability of DDX1 to promote oligomerization of Rev on the RRE may be a general property shared by other DEAD-box helicases, or this activity might reflect the helicase activities and substrate preferences of a particular helicase.…”

“…Therefore, only an assay that directly measures the binding of Rev to the RRE can be used to assess effects of DDX proteins on Rev binding and subsequent oligomerization on the RRE. One such method uses total internal reflection fluorescence (TIRF) microscopy to visualize Rev association with the RRE at single molecule resolution[14, 23, 24, 46]. In this experiment, a full length RRE molecule is immobilized on a quartz surface via biotin-streptavidin attachment, utilizing a biotinylated oligonucleotide that is hybridized at the 3′ end of the RRE.…”

“…In this study, the RNA-dependent ATPase, RNA binding, and protein binding activities of DDX21 are compared to the previously published results for DDX1 and DDX3 [13, 23, 35]. Further, these activities are discussed in the context of the Rev:RRE regulatory pathway, including the effect of each helicase on Rev oligomerization on the RRE.…”

“…Two of these DEAD-box helicases, DDX1 and DDX3, had been previously implicated in the HIV-1 Rev regulatory pathway[10–22]. DDX1 directly interacts with Rev and RCCs, playing a role in the correct localization of Rev within the nucleus in vivo , as well as affecting Rev’s association with and oligomerization on the RRE in vitro [10, 12, 14, 15, 23, 24]. DDX3 affects HIV production at the mRNA export, protein translation and budding steps of the viral lifecycle, though what role, if any, Rev may play in these processes remains unclear[10, 18, 19, 21].…”