Dimerization Initiation Site Research Articles

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.

Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes

HIV-1 RNA dimerization is a critical step in viral life cycle. It is a prerequisite for genome packaging and plays an important role in reverse transcription and recombination. Dimerization is promoted by the DIS (dimerization initiation site) hairpin located in the 5′ leader of HIV-1 genome. Despite the high genetic diversity in HIV-1 group M, only five apical loops (AAGCGCGCA, AAGUGCGCA, AAGUGCACA, AGGUGCACA and AGUGCAC) are commonly found in DIS hairpins. We refer to the parent DISes with these apical loops as DISLai, DISTrans, DISF, DISMal, and DISC, respectively. Based on identity or similarity of DIS hairpins to parent DISes, we distributed HIV-1 M genomes into five dimerization groups. Comparison of the primary and secondary structures of DIS, SD and Psi hairpins in about 3000 HIV-1 M genomes showed that the mutation frequencies at particular nucleotide positions of these hairpins differ among the dimerization groups, and DISF may be an origin of other parent DISes. We found that DIS, SD and Psi hairpins have hundreds of variants, only some of them occurring rather frequently. The lower part of DIS hairpin with G x AGG internal loop is highly conserved in both HIV-1 and SIV genomes. We supposed that the G-quadruplex, located 56 nts downstream of the Gag start codon, may participate in switching of HIV-1 leader RNA from BMH (branched multiple hairpins) to LDI (long distance interaction) conformation.

Selective packaging of HIV-1 RNA genome is guided by the stability of 5′ untranslated region polyA stem

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

B-CePs as cross-linking probes for the investigation of RNA higher-order structure.

Elucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane.

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

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.

Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation.

A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.

Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.

Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes.

Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.

Intrinsic conformational dynamics of the HIV-1 genomic RNA 5′UTR

The highly conserved 5' untranslated region (5'UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5'UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5'UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5'UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3 Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

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.

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.

Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach

Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25kDa, respectively. We combined these techniques to study a 30kDa HIV-1 dimer initiation site RNA ([DIS]2; 47nt/strand). A 9Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.

586. Dimerization Inhibition of HIV-1 RNA by 2’-Deoxy-2’-fluoro-beta-D-arabinose Nucleic Acid (2’-FANA) Modified Antisense Oligonucleotides Results in Potential Inhibition of Viral Expression

Human immunodeficiency virus-1 (HIV-1) viral particle contains two copies of genomic RNA, which is known to form dimers via intermolecular interactions. The dimerization process is initiated by the formation of a kissing-loop dimer through base pairing of the palindromic loop sequence within dimerization initiation site (DIS). It has been shown that mutation or inhibitions of the DIS severely affect the viral infectivity. Antisense oligonucleotides (AONs) are single-stranded synthetic oligonucleotides that recognize target RNAs via Watson-Crick base pairing and cause post-translational inhibition. The mechanisms are believed to be RNase H cleavage of target RNA, steric hindrance of the translation machinery or prevention of RNA-RNA or RNA-protein interactions. While AONs offer promising solutions for variety of human diseases in preclinical studies and many of these are currently in clinical studies, a number of challenges still hamper their translation from the bench to the bedside, the most significant of which include target accessibility, off target effects, poor extracellular and intracellular stability and effective delivery into target cells. 2’-deoxy-2’-fluoro-beta-D-arabinose nucleic acid (2’-FANA) modification significantly enhances chemical and intracellular stability, as well as binding to the target RNA, forming stable heteroduplex structures. Moreover, 2’-FANA modification has been shown to facilitate gymnotic cellular delivery, a carrier-free method of achieving cellular uptake. Thus, we hypothesized that 2’-FANA modified AONs that bind HIV-1 DIS can inhibit dimerization, and subsequently inhibit viral expression. Firstly, we tested gymnotic delivery of 2’-FANA modified AONs to peripheral blood mononuclear cells (PBMCs) freshly isolated from human blood. Cellular uptake of the 2’-FANA AONs without any transfection reagent were observed in live-cell confocal microscopy analysis as soon as an hour after adding AONs. Next, the HIV-1 inhibitory effect of the 2’-FANA AONs was tested in HIV-1 infected PBMCs. Cell supernatant was collected and HIV-1 expression was measured by HIV-1 p24 ELISA. The 2’-FANA AONs strongly inhibited HIV-1 expression as long as two weeks after 2’-FANA AONs treatment. A mechanism study showed that the 2’-FANAAONs work against HIV by steric blocking of dimerization. Unlike most oligonucleotide therapeutics, 2’-FANA AONs can be gymnotically delivered into PBMCs. This fact makes 2’-FANA AONs great drug candidates for antiretroviral therapy.

CHARACTERIZATION OF THE HIV‐1 5′UTR DIMERIZATION MECHANISM

Human Immunodeficiency Virus Type‐1 (HIV‐1) is responsible for a pandemic that affects roughly 35 million people worldwide. To date, there is no cure, and the array of antiretroviral medications serves only to diminish the progression of the disease and prevent a more rapid deterioration of the immune system. Many antiretrovirals target four main stages of the viral life cycle: entry, reverse transcription, integration, and maturation. Genome selection for packaging, however, is relatively less clearly understood, thus no current therapies that target this process have been developed. HIV‐1 genome packaging has been shown to be a highly selective process in which only dimeric, unspliced, genomes are recognized by the nucleocapsid (NC) domain of the Gag polyprotein for encapsidation. The nature of dimerization, which is mediated by elements within the 5′ untranslated region (5′‐UTR) of the genome, is hypothesized to be either a “kissing” interaction involving the six nucleotide palindromic (GCGCGC) dimerization initiation site (DIS) sequence, or an extended dimer conformation featuring a more extensive intermolecular interface. Our studies aim to elucidate the dimerization mechanism using NMR spectroscopy. Previous studies in the Summers laboratory identified that the HIV‐1 5′‐UTR exists as an extended dimer at high concentrations and after long incubation times at 37 °C. The U5:AUG region, which is distal to the DIS, has been identified as one of the sites with an intermolecular interface. We hypothesize that dimer formation is initiated by kissing interactions and transitions to an extended conformation over time. We utilized NMR spectroscopy to test this hypothesis using a mutagenesis strategy know as long‐range Adenosine Interaction Detection (lr‐AID). 1D NMR data collected on small U5:AUG control RNAs containing either UUA/UAA or UUG/UAA lr‐AID signals revealed isolated chemical shifts at 6.5 ppm, and 6.7 ppm, respectively. We incorporated the UUA/UAA and the UUG/UAA sequences within the U5:AUG region of the 5′‐UTR and observed the appearance of a chemical shift at 6.5 ppm in a biologically relevant time frame. Currently, we are conducting time‐dependent dimerization studies using native gel electrophoresis techniques in order to further characterize this transition.Support or Funding InformationThis research was funded by NIH/NIGMS grant 1P50GM103297 and sponsored by NIH/NIGMS MARC U*STAR T34 08663 National Research Service award to UMBC. It was conducted at the Howard Hughes Medical Institute at UMBC, with support from the Summer Biomedical Training Program (SBTP). Supported in part by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program.

New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging.

Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

A Conserved Mechanism within the 5'-Leader RNA Genome among HIV-1 and SIVcpz Strains during Viral Genome Recognition

Previous studies in our lab suggested that HIV-1 5′-Leader (5′-L) RNA genome is regulated by a molecular structural switch. The dimer promoting GC-rich loop of the dimer initiation site (DIS) hairpin is sequestered by base pairing with the unique-5’ region (U5) in the monomeric form. The DIS:U5 interaction is then displaced by a stem-loop region containing gag start codon (AUG). The U5:AUG interaction, therefore, occurs and promotes dimerization of the 5′-L.The question raised is whether this structural switch conserved in lentivirus family. Sequence alignment, gel electrophoresis, ITC, and NMR methods are employed in this study to compare RNA constructs from HIV-1, SIVcpz_TAN1, and SIVcpz_US strains. We found both SIVcpz_TAN1 and US strains utilize a similar RNA structural switch to HIV-1 within their 5′-L. This conserved function can be a potential candidate for anti-viral drug development.

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.

SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

Earlier genetic and structural prediction analyses revealed that the packaging determinants of Mason Pfizer monkey virus (MPMV) include two discontinuous core regions at the 5' end of its genomic RNA. RNA secondary structure predictions suggested that these packaging determinants fold into several stem-loops (SLs). To experimentally validate this structural model, we employed selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), which examines the flexibility of the RNA backbone at each nucleotide position. Our SHAPE data validated several predicted structural motifs, including U5/Gag long-range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive G-C-rich palindromic (pal) SL. Minimum free-energy structure predictions, phylogenetic, and in silico modeling analyses of different MPMV strains revealed that the U5 and gag sequences involved in the LRIs differ minimally within strains and maintain a very high degree of complementarity. Since the pal SL forms a helix loop containing a canonical "GC" dyad, it may act as a RNA dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Analyses of wild-type and pal mutant RNAs revealed that disruption of pal sequence strongly affected RNA dimerization. However, when in vitro transcribed trans-complementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5'-CGGCCG-3') functions as DIS.