gRNA Dimerization Research Articles

Mapping the structural landscape of the yeast Ty3 retrotransposon RNA genome.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5' ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.

Determinants of the interaction between the 5′-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3′ end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5′ leader (5′L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5′L responsible for LysRS binding, how the conformations and various hairpin structures of 5′L regulate 5′L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5′L significantly influences its binding affinity with LysRS. The 5′L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5′L conformation that is not selected for packaging. Additionally, dimerization of 5′L impairs LysRS-5′L interaction. Furthermore, among various regions of 5′L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5′L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

Theory of HIV-1 RNA genome selection.

It is not understood how the HIV-1 retrovirus selects it genomic RNA (gRNA) for packaging into virions from among the multitude of cellular RNA molecules in the cytoplasm of infected cells. Under in-vitro conditions, the Gag proteins of HIV-1 have only a very weak specific binding affinity for the RNA Ψ sequence, which has been shown to be the key recognition label of genomic RNA. In addition, the cellular gRNA concentration is very low yet HIV packages gRNA with high fidelity. We present a numerical model for kinetic HIV-1 gRNA selection that is based on fusion of precursor buds followed by gRNA dimerization.

Distinct Gag interaction properties of HIV-1 RNA 5' leader conformers reveal a mechanism for dimeric genome selection.

During HIV-1 assembly, two copies of viral genomic RNAs (gRNAs) are selectively packaged into new viral particles. This process is mediated by specific interactions between HIV-1 Gag and the packaging signals at the 5' leader (5'L) of viral gRNA. 5'L is able to adopt different conformations, which promotes either gRNA dimerization and packaging or Gag translation. Dimerization and packaging are coupled. Whether the selective packaging of the gRNA dimer is due to favorable interactions between Gag and 5'L in the packaging conformation is not known. Here, using RNAs mimicking the two 5'L conformers, we show that the 5'L conformation dramatically affects Gag-RNA interactions. Compared to the RNA in the translation conformation (5'LT), the RNA in the packaging conformation (5'LP) can bind more Gag molecules. Gag associates with 5'LP faster than it binds to 5'LT, whereas Gag dissociates from 5'LP more slowly. The Gag-5'LP complex is more stable at high salt concentrations. The NC-SP2-p6 region of Gag likely accounts for the faster association and slower dissociation kinetics for the Gag-5'LP interaction and for the higher stability. In summary, our data suggest that conformational changes play an important role in the selection of dimeric genomes, probably by affecting the binding kinetics and stability of the Gag-5'L complex.

RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation.

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC.

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag.

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA

ABSTRACTDuring replication of long terminal repeat (LTR)-retrotransposons, their proteins and genome (g) RNA assemble into virus-like particles (VLPs) that are not infectious but functionally related to retroviral virions. Both virions and VLPs contain gRNA in a dimeric form, but contrary to retroviruses, little is known about how gRNA dimerization and packaging occurs in LTR-retrotransposons. The LTR-retrotransposon Ty1 from Saccharomyces cerevisiae is an informative model for studying LTR-retrotransposon and retrovirus replication. Using structural, mutational and functional analyses, we explored dimerization of Ty1 genomic RNA. We provide direct evidence that interactions of self-complementary PAL1 and PAL2 palindromic sequences localized within the 5′UTR are essential for Ty1 gRNA dimer formation. Mutations disrupting PAL1-PAL2 complementarity restricted RNA dimerization in vitro and Ty1 mobility in vivo. Although dimer formation and mobility of these mutants was inhibited, our work suggests that Ty1 RNA can dimerize via alternative contact points. In contrast to previous studies, we cannot confirm a role for PAL3, tRNAiMet as well as recently proposed initial kissing-loop interactions in dimer formation. Our data also supports the critical role of Ty1 Gag in RNA dimerization. Mature Ty1 Gag binds in the proximity of sequences involved in RNA dimerization and tRNAiMet annealing, but the 5′ pseudoknot in Ty1 RNA may constitute a preferred Gag-binding site. Taken together, these results expand our understanding of genome dimerization and packaging strategies utilized by LTR-retroelements.

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

ABSTRACTPackaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Probing RNA Structure and Dynamics in the HIV‐1 5′ UTR using Ensemble and Single Molecule Approaches

One of the most conserved and highly structured regions of the HIV‐1 genome, the 356‐nt 5′UTR, acts as a scaffold for many protein and nucleic acid binding partners and regulates vital processes including initiation of reverse transcription, genomic RNA (gRNA) dimerization and packaging. Previous studies support the presence of at least two mutually‐exclusive 5′UTR conformational states (Huthoff and Berkhout, RNA, 7, 2001; Lu et al., Science 334 (2011), but whether the 5′UTR undergoes a conformational switch during the viral lifecycle and what regulates this switch are less clear. In‐gel selective 2′‐hydroxyl acylation followed by primer extension (SHAPE) probing studies of the wild‐type (WT) 5′UTR revealed a mixed population with structural differences between monomer and dimer, consistent with a structural switch (Kenyon, JC et al, Nucleic Acids Res 41, 2013). However, these studies did not address the dynamics of the conformational change or probe possible triggers. In this work, we used single‐molecule Förster resonance energy transfer (SM‐FRET) studies to probe 5′UTR (ΔTAR/polyA) conformational dynamics and the factors that modulate it. Intra‐molecular FRET assays showed that mutations in the dimerization initiation site (DIS) and U5 region of the monomeric RNA modulated the conformational equilibrium in the expected manner, with the ΔDIS construct shifted to a high‐FRET dimerization‐competent state. Increasing Mg2+ concentration promoted the dimer‐competent conformation in the WT 5′UTR. In 10 mM Mg2+, the WT construct occupies both the high‐FRET (39%) and low‐FRET states (61%). Individual FRET traces showed that of the 841 molecules examined under these conditions, 29% transitioned between FRET states during the ~150 sec observation window. Inter‐molecular FRET observed between an immobilized donor‐labeled 5′UTR construct and excess acceptor‐labeled 5′UTR was consistent with formation of a stable extended dimer. In‐gel FRET assays were also consistent with the formation of a stable dimer. Surprisingly, tRNA‐annealing to the primer binding site of an immobilized 5′UTR promoted the monomer conformation even in the presence of high Mg2+, as observed by both SM‐FRET and native PAGE. Overall, these data are consistent with high intrinsic conformational dynamics in the 5′UTR that can be regulated by factor binding.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Retroviral RNA Dimerization: From Structure to Functions.

The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…), the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry

ABSTRACTThe HIV-1 Pr55Gag precursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55Gag and a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55Gag specifically associate with the 5′ region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55Gag binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55Gag. Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55Gag recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55Gag among a variety of svRNAs, all harboring SL1 in their first common exon.

Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV).

BackgroundOne of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5’ end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5’ untranslated region (5’ UTR) and the beginning of gag.ResultsThe RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2’hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5’ region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5’ CGGCCG 3’) at the 5’ UTR and contains a canonical “GC” dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging.ConclusionsEmploying structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0096-6) contains supplementary material, which is available to authorized users.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor.

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

Moloney murine leukemia virus genomic RNA packaged in the absence of a full complement of wild type nucleocapsid protein

The current model for MLV genomic RNA (gRNA) packaging predicts that of the thousands of Gag proteins in a budding virion, only a small number (≤1%) may be necessary to recruit gRNA. Here, we examined the threshold limits of functional Gag required to package gRNA using wild-type (WT) and packaging deficient mutant nucleocapsid (NC) phenotypically mixed virions. Although gRNA packaging was severely diminished for the NC mutant, the residual encapsidated RNA dimer displayed motility on gels, thermostability, and integrity that was indistinguishable from that of WT. In phenotypically mixed virions, gRNA encapsidation recovered to within approximately two-fold of WT levels when the amount of WT NC was 5–10% of the total. Our results demonstrate that NC's roles in gRNA dimerization and packaging are genetically separable. Additionally, MLV gRNA packaging does not require 100% WT NC, but the amount of functional NC required is greater than the predicted minimum.

Dominant Role of the 5′ TAR Bulge in Dimerization of HIV-1 Genomic RNA, but No Evidence of TAR–TAR Kissing during in Vivo Virus Assembly

The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The ΔUCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than ΔUCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.

Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: Differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site

Formation of immature genomic RNA (gRNA) dimers is exquisitely nucleocapsid (NC)-dependent in protease-inactive (PR-in) HIV-1. This establishes that Pr55gag/Pr160gag-pol has NC-dependent chaperone activity within intact HIV-1. Mutations in the proximal zinc finger and the linker of the NC sequence of Pr55gag/Pr160gag-pol abolish gRNA dimerization in PR-in HIV-1. In wild type, where the NC of Pr55gag is processed into progressively smaller proteins termed NCp15 (NCp7-p1-p6), NCp9 (NCp7-p1) and NCp7, formation of immature dimers is much swifter than in PR-in HIV-1. NCp7 and NCp15 direct this rapid accumulation. NCp9 is sluggish in this process, but it stimulates the transition from immature to mature gRNA dimer as well as NCp7 and much better than NCp15. The amino-terminus, proximal zinc finger, linker, and distal zinc finger of NCp7 contribute to this maturation event in intact HIV-1. The DIS is a dimerization initiation site for all immature gRNA dimers, irrespective of their mechanism of formation.

Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

HIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7–p1–p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7–p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1–p6) and tertiary (p7–p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7–p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization.

Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging

Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity, and the nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) facilitates HIV-1 gRNA dimerization. To identify the relevant and dispensable positions of NC, 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants prepared by site-directed mutagenesis. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The N-terminal and linker segments appeared to play predominantly electrostatic and steric roles, respectively. Mutating the hydrophobic patch of either zinc finger, or substituting alanines for their glycine doublet, was as disabling as deleting the corresponding finger. Replacing the CysX2CysX4HisX4Cys motif of either finger by CysX2CysX4CysX4Cys or CysX2CysX4HisX4His, interchanging the zinc fingers or, replacing one zinc finger by a copy of the other one, had generally intermediate effects; among these mutations, the His23→Cys substitution in the N-terminal zinc finger had the mildest effect. The charge of NC could be increased or decreased by up to 18%, that of the linker could be reduced by 75% or increased by 50%, and one or two electric charges could be added or subtracted from either zinc finger, without affecting gRNA dimerization. Shortening, lengthening, or making hydrophobic the linker was as disabling as deleting the N-terminal or the C-terminal zinc finger, but a neutral and polar linker was innocuous. The present work multiplies by 4 and by 33 the number of retroviral and lentiviral NC mutations known to inhibit gRNA dimerization, respectively. It shows the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein. Most, but not all, mutations inhibited gRNA packaging; some had a strong effect on virus assembly or stability.