Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

Abstract

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5= untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10-to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCETo generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5= end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts.

RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

“…The deletion landscape for HIV-1 recapitulated known CAEs (LTR, Y, RRE) and their roles in transcription, encapsidation and egress. Despite previous claims that the Genomic RNA Packaging Enhancer (GRPE) is important for encapsidation (40), deletions of this region (nucleotide position 2022-2188) did not affect mobilization, in agreement with the findings of Nikolaitchik and Hu (72). The results also showed that the cPPT, despite its debated role in HIV-1 replication, was as important for sustained viral replication as the LTR, Y, and RRE.…”

Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA

“…We have analyzed the binding of Gag to three 175-base viral-derived RNAs: HIV-1 nt 193–368, referred to here as ‘Ψ'; HIV-1 nt 2004–2179, called ‘GRPE’; and Moloney MLV nt 202–377, called ‘MoMLV Ψ'. GRPE has been suggested to contribute to selective packaging of gRNA ( Chamanian et al, 2013 ), but this now seems unlikely ( Nikolaitchik et al, 2014 ). As many lines of evidence indicate that only dimeric gRNA is selectively packaged ( Moore et al, 2009 ), we studied the HIV-1 Ψ RNA in both monomeric and dimeric forms (see Materials and methods).…”

HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus

“…When ΔZF2 virions were co-assembled with ΔZF2Gag c -Tsg101, the DNA content of the trans -complemented virions decreased while the gRNA level increased. Deleting the ZF2 sequence could also generate a cis -effect at the RNA level by inactivating the RNA packaging enhancer signal (named GRPE), which overlaps with the NC ZF2 sequence ( 47 ), although this role remains controversial ( 48 ). Thus, in this case the ΔZF2 gRNA molecules could not be packaged as efficiently as the WT gRNA and this could explain why a complete rescue of ΔZF2 was not achieved by Tsg101.…”