Conserved determinants of lentiviral genome dimerization.

Thao Tran,Mateo Hernandez,Alexander Vega,Jennifer Bohn,Jan Marchant,Michael F Summers,Venkateswaran Ramakrishnan,Yuanyuan Liu,Sarah Monti,Jessica Zaki,Ae Lim Yang,Michelle Seu,Rashmi Singh

doi:10.1186/s12977-015-0209-x

Thao Tran, Mateo Hernandez + Show 11 more

Open Access

https://doi.org/10.1186/s12977-015-0209-x

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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.

Highlights

Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism
RNA construct design Full-length, wild-type constructs (5′-LWT) for HIV-1NL4-3, SIVcpzTAN1, SIVcpzUS, and HIV-2ROD viral strains were designed to include all residues required for the formation of the proposed 3′-terminal AUG (Fig. 1a) [33, 51]
Non‐native 3′‐residues influence the dimerization behavior of the HIV‐1 5′ leader Time-dependent RNA dimerization data were obtained by dissolution of a concentrated buffer/salt solution into HIV-1NL4-3 5′-LWT solutions that were pre-incubated at low ionic strength, such that the final solutions contained physiological-like ionic conditions (PI buffer; 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM Tris–HCl, pH 7.0)

Summary

Introduction

Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. NMR studies confirmed the presence of U5:AUG base pairing in the dimeric form of the HIV-1 leader RNA [33], and U5:AUG formation was further shown to promote both dimerization and binding to the cognate nucleocapsid (NC) protein in vitro, as well as selective and efficient packaging of vector RNAs into virus-like particles in transfected cell cultures [33] These and other findings suggested packaging mechanisms in which a U5:AUG dependent RNA structural switch leads to the formation of structures that expose both the dimer initiation site (DIS) and high affinity NC binding sites, thereby promoting selective packaging of the dimeric genome [2, 13, 21, 33, 36]

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