Abstract
The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.
Highlights
Short purine-rich segments were mutated that flank the dimerization initiation signal (DIS) stem on the 5Ј side (240GGA242 triplet mutated to CCC) and 3Ј side (278GGG280 to UCC) in J8 and J9 to extend the DIS stem with three additional base pairs (Fig. 2A)
A dimeric RNA template is likely required to facilitate the complex and nonlinear reverse transcription process that generates a DNA copy that is longer than the original RNA template
The dimeric RNA genome may allow the virus to bypass nicks or lesions in one of the RNA strands [65, 66], and it may be beneficial for virus evolution by inducing recombination events [67, 68]
Summary
Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, indicating that the ED structure is important for HIV-1 replication. Heat treatment or incubation with the viral nucleocapsid protein triggers opening of the hairpin stem to allow extended interstrand base pairing and formation of a more stable extended dimer (ED) (Fig. 1, B and C) (9 –11) Such ED RNA forms have only been described in vitro, and their in vivo relevance remains unclear. Moore et al [24] created DIS mutants that, at least in vitro, can form KL dimers, but not ED Their experiments indicated that ED formation is not required for the initial RNA partner selection, subsequent RNA packaging, and the process of recombination. Characterization of these variants confirmed the importance of the GGA triplet in both ED formation and viral replication
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