A Riboswitch Regulates RNA Dimerization and Packaging in Human Immunodeficiency Virus Type 1 Virions

Abstract

The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the ⌿ and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the ⌿ hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies ( This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.Retroviral particles package a dimeric RNA genome that is subsequently reverse transcribed into DNA by the virion-associated reverse transcriptase enzyme. The cis-acting RNA sequences and the trans-acting viral proteins that mediate RNA dimerization and RNA packaging have been studied extensively for several retroviruses, including human immunodeficiency virus type 1 (HIV-1) (9,20,35). It has proven particularly difficult to accurately map the RNA signals that execute these two processes, but most studies have indicated that these elements cluster within the untranslated leader region of the HIV-1 genome. In vitro studies have identified the dimerization initiation signal (DIS) as the primary dimerization signal, which acts through base pairing of a palindromic 6-nucleotide (nt) sequence motif in the exposed loop of the DIS hairpin (4,27,32,40). Nevertheless, studies with mutant virions have indicated that the in vivo situation is much more complex, suggesting the possibility of multiple accessory dimerization signals (6,21,38). The ⌿ hairpin has been implicated as the core element that mediates packaging, but there is ample evidence for the involvement of additional sequence elements (25,28). It has also been suggested that the process of RNA dimerization may be coupled to RNA encapsidation; such a coupling seems to be an elegant mechanism for ensuring that only dimeric RNA genomes are packaged (18).Huthoff and Berkhout demonstrated that the HIV-1 untranslated leader RNA can ado...

Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA

“…15,68,69 These types of conformational switches are important for HIV-1 and MLV, for example, in the encapsidation process. [70][71][72] Furthermore, one major finding suggested that a fraction of the gag mRNA (vRNA) is retained in distinct cytoplasmic compartments in both the absence and presence of Rev. 17 Our results confirm this finding and also reveal how cellular proteins, like hnRNP A2/B1, influence the dynamic formation of these HIV-1 RNA pools by influencing tightly regulated nuclear and cytoplasmic trafficking events.…”

HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms

“…polyA and SD hairpin; Das et al 1997Das et al , 1999aAbbink & Berkhout 2007), which eventually led to the description of an alternative HIV-1 RNA leader structure in which the polyA hairpin is opened to allow base-pairing with the DIS domain (Huthoff & Berkhout 2001;Abbink & Berkhout 2003;Ooms et al 2004). The equilibrium between the two leader conformations may function as a riboswitch that is important in controlling RNA dimerization and packaging.…”

Cell biology of retroviral RNA packaging

“…Such RNA conformational switches have been elucidated upon dimerization of HIV-1 and MLV RNAs. 11,12,19,[93][94][95][96][97][98][99] However, direct insights into RNA structure-function relationship are needed to validate this hypothesis. Another possibility is that the interaction between the gRNA and Gag, or between the gRNA and a-yet-to-be-identified cellular factor, could modulate the dual targeting of the genome.…”