Conserved determinants of lentiviral genome dimerization

Abstract

BackgroundRetroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by “kissing” interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5′ element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5′ leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy.ResultsDimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg2+, indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers.ConclusionsThe finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5′ leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

“…Surprisingly, the influence of the 5′-Cap moiety on dimerization was similar to that of adding a single phosphodiester-linked 5′ guanosine. Our findings support proposals that genome dimerization is a major determinant of RNA function (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(25)(26)(27)(28)(29)(30)(31)(32)(33) and suggest a paradigm in which the structure, function, and fate of the viral transcripts are modulated by heterogeneous TSS selection.…”

“…Because prior studies have established that genome dimerization likely controls packaging and other RNA-dependent functions (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(25)(26)(27)(28)(29)(30)(31)(32)(33), we examined the influence of TSS use on the in vitro dimerization properties of the HIV-1 NL4-3 5′-L (Fig. 4A).…”

“…To identify molecular determinants of retroviral RNA structure, we recently examined the dimerization properties of native and mutant 5′-L RNAs of several lentiviruses (31). Unexpectedly, RNAs reported to start with three 5′ guanosines exhibited dimerization properties substantially different from those that begin with two guanosines.…”

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

“…1; Jaballah et al 2010;Aktar et al 2013). LRIs have been found to be conserved in several retroviruses, including HIV-1 and 2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and mouse mammary tumor virus (MMTV) (Paillart et al 2002(Paillart et al , 2004bAbbink and Berkhout 2003;Kenyon et al 2008Kenyon et al , 2011Aktar et al 2014;Siegfried et al 2014;Keane et al 2015;Smyth et al 2015;Tran et al 2015). Despite the fact that there are substantial sequence heterogeneities among human, simian, and feline lentiviruses, the preservation of such LRIs in these lentiviruses suggest they play important function(s) in the retroviral life cycle (Paillart et al 2002;Kenyon et al 2008Kenyon et al , 2011.…”

HIV-1 leader RNA dimeric interface revealed by NMR

“…This conformational switch was further supported by NMR data showing that NMR signals of AUG in the monomeric leader match those of an isolated AUG hairpin, and AUG signals in the dimeric leader match those of an isolated AUG-U5 duplex, and by experiments showing that mutations that inhibit or promote these interactions have the expected effects on dimerization and packaging (18). Recent studies on HIV-2 and simian immunodeficiency virus suggest that this molecular switch is highly conserved among retroviruses (19). Additional mutational studies helped define a minimal core encapsidation signal (Ψ CES ) within the leader that retains the key structural features of the dimer, as well as elements needed to direct packaging (Fig.…”