Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Sequestering the 5'-cap for viral RNA packaging.

Many viruses evolved mechanisms for capping the 5'-ends of their plus-strand RNAs as a means of hijacking the eukaryotic messenger RNA (mRNA) splicing/translation machinery. Although capping is critical for replication, the RNAs of these viruses have other essential functions including their requirement to be packaged as either genomes or pre-genomes into progeny viruses. Recent studies indicate that human immunodeficiency virus type-1 (HIV-1) RNAs are segregated between splicing/translation and packaging functions by a mechanism that involves structural sequestration of the 5'-cap. Here, we examined studies reported for other viruses and retrotransposons that require both selective packaging of their RNAs and 5'-RNA capping for host-mediated translation. Our findings suggest that viruses and retrotransposons have evolved multiple mechanisms to control 5'-cap accessibility, consistent with the hypothesis that removal or sequestration of the 5' cap enables packageable RNAs to avoid capture by the cellular RNA processing and translation machinery.

5'-Cap sequestration is an essential determinant of HIV-1 genome packaging.

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

HIV-1 matrix-tRNA complex structure reveals basis for host control of Gag localization.

The HIV-1 virion structural polyprotein, Gag, is directed to particle assembly sites at the plasma membrane by its N-terminal matrix (MA) domain. MA also binds to host tRNAs. To understand the molecular basis of MA-tRNA interaction and its potential function, we present a co-crystal structure of HIV-1 MA-tRNALys3 complex. The structure reveals a specialized group of MA basic and aromatic residues preconfigured to recognize the distinctive structure of the tRNA elbow. Mutational, cross-linking, fluorescence, and NMR analyses show that the crystallographically defined interface drives MA-tRNA binding in solution and living cells. The structure indicates that MA is unlikely to bind tRNA and membrane simultaneously. Accordingly, single-amino-acid substitutions that abolish MA-tRNA binding caused striking redistribution of Gag to the plasma membrane and reduced HIV-1 replication. Thus, HIV-1 exploits host tRNAs to occlude a membrane localization signal and control the subcellular distribution of its major structural protein.

Correction: Boyd, P.S., et al. NMR Studies of Retroviral Genome Packaging. Viruses 2020, 12, 1115.

There was an error in the original article [...].

NMR Studies of Retroviral Genome Packaging.

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems-a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal.

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 µM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.

Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus.

All retroviruses encode a Gag polyprotein containing an N-terminal matrix domain (MA) that anchors Gag to the plasma membrane and recruits envelope glycoproteins to virus assembly sites. Membrane binding by the Gag protein of HIV-1 and most other lentiviruses is dependent on N-terminal myristoylation of MA by host N-myristoyltransferase enzymes (NMTs), which recognize a six-residue "myristoylation signal" with consensus sequence: M1GXXX[ST]. For unknown reasons, the feline immunodeficiency virus (FIV), which infects both domestic and wild cats, encodes a non-consensus myristoylation sequence not utilized by its host or by other mammals (most commonly: M1GNGQG). To explore the evolutionary basis for this sequence, we compared the structure, dynamics, and myristoylation properties of native FIV MA with a mutant protein containing a consensus feline myristoylation motif (MANOS) and examined the impact of MA mutations on virus assembly and ability to support spreading infection. Unexpectedly, myristoylation efficiency of MANOS in Escherichia coli by co-expressed mammalian NMT was reduced by ~70% compared to the wild-type protein. NMR studies revealed that residues of the N-terminal myristoylation signal are fully exposed and mobile in the native protein but partially sequestered in the MANOS chimera, suggesting that the unusual FIV sequence is conserved to promote exposure and efficient myristoylation of the MA N terminus. In contrast, virus assembly studies indicate that the MANOS mutation does not affect virus assembly, but does prevent virus spread, in feline kidney cells. Our findings indicate that residues of the FIV myristoylation sequence play roles in replication beyond NMT recognition and Gag-membrane binding.

Structural basis for transcriptional start site control of HIV-1 RNA fate.

Heterogeneous transcriptional start site usage by HIV-1 produces 5'-capped RNAs beginning with one, two, or three 5'-guanosines (Cap1G, Cap2G, or Cap3G, respectively) that are either selected for packaging as genomes (Cap1G) or retained in cells as translatable messenger RNAs (mRNAs) (Cap2G and Cap3G). To understand how 5'-guanosine number influences fate, we probed the structures of capped HIV-1 leader RNAs by deuterium-edited nuclear magnetic resonance. The Cap1G transcript adopts a dimeric multihairpin structure that sequesters the cap, inhibits interactions with eukaryotic translation initiation factor 4E, and resists decapping. The Cap2G and Cap3G transcripts adopt an alternate structure with an elongated central helix, exposed splice donor residues, and an accessible cap. Extensive remodeling, achieved at the energetic cost of a G-C base pair, explains how a single 5'-guanosine modifies the function of a ~9-kilobase HIV-1 transcript.

Biochemical Reconstitution of HIV-1 Assembly and Maturation.

The assembly of an orthoretrovirus such as HIV-1 requires the coordinated functioning of multiple biochemical activities of the viral Gag protein. These activities include membrane targeting, lattice formation, packaging of the RNA genome, and recruitment of cellular cofactors that modulate assembly. In most previous studies, these Gag activities have been investigated individually, which provided somewhat limited insight into how they functionally integrate during the assembly process. Here, we report the development of a biochemical reconstitution system that allowed us to investigate how Gag lattice formation, RNA binding, and the assembly cofactor inositol hexakisphosphate (IP6) synergize to generate immature virus particles in vitro The results identify an important rate-limiting step in assembly and reveal new insights into how RNA and IP6 promote immature Gag lattice formation. The immature virus-like particles can be converted into mature capsid-like particles by the simple addition of viral protease, suggesting that it is possible in principle to fully biochemically reconstitute the sequential processes of HIV-1 assembly and maturation from purified components.IMPORTANCE Assembly and maturation are essential steps in the replication of orthoretroviruses such as HIV-1 and are proven therapeutic targets. These processes require the coordinated functioning of the viral Gag protein's multiple biochemical activities. We describe here the development of an experimental system that allows an integrative analysis of how Gag's multiple functionalities cooperate to generate a retrovirus particle. Our current studies help to illuminate how Gag synergizes the formation of the virus compartment with RNA binding and how these activities are modulated by the small molecule IP6. Further development and use of this system should lead to a more comprehensive understanding of the molecular mechanisms of HIV-1 assembly and maturation and may provide new insights for the development of antiretroviral drugs.

Assigning NMR spectra of RNA, peptides and small organic molecules using molecular network visualization software.

NMR assignment typically involves analysis of peaks across multiple NMR spectra. Chemical shifts of peaks are measured before being assigned to atoms using a variety of methods. These approaches quickly become complicated by overlap, ambiguity, and the complexity of correlating assignments among multiple spectra. Here we propose an alternative approach in which a network of linked peak-boxes is generated at the predicted positions of peaks across all spectra. These peak-boxes correlate known relationships and can be matched to the observed spectra. The method is illustrated with RNA, but a variety of molecular types should be readily tractable with this approach.

Synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of HIV-1.

Anti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.

Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein.

NMR has provided a wealth of structural and dynamical information for RNA molecules of up to ∼50 nucleotides, but its application to larger RNAs has been hampered in part by difficulties establishing global structural features. A potential solution involves measurement of NMR perturbations after site-specific paramagnetic labeling. Although the approach works well for proteins, the inability to place the label at specific sites has prevented its application to larger RNAs transcribed in vitro. Here, we present a strategy in which RNA loop residues are modified to promote binding to a paramagnetically tagged reporter protein. Lanthanide-induced pseudocontact shifts are demonstrated for a 232-nucleotide RNA bound to tagged derivatives of the spliceosomal U1A RNA-binding domain. Further, the method is validated with a 36-nucleotide RNA for which measured NMR values agreed with predictions based on the previously known protein and RNA structures. The ability to readily insert U1A binding sites into ubiquitous hairpin and/or loop structures should make this approach broadly applicable for the atomic-level study of large RNAs.

Evaluation of global HIV/SIV envelope gp120 RNA structure and evolution within and among infected hosts.

Lentiviral RNA genomes contain structural elements that play critical roles in viral replication. Although structural features of 5'-untranslated regions have been well characterized, attempts to identify important structures in other genomic regions by Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) have led to conflicting structural and mechanistic conclusions. Previous approaches accounted neither for sequence heterogeneity that is ubiquitous in viral populations, nor for selective constraints operating at the protein level. We developed an approach that augments SHAPE with phylogenetic analyses and applied it to investigate structure in coding regions (cRNA) within the HIV and SIV envelope genes. Analysis of single-genome SHAPE data with phylogenetic information from diverse lentiviral sequences argues against the conservation of a putative global gp120 RNA structure but points to the existence of core RNA sub-structures. Our findings establish a framework for considering sequence heterogeneity and protein function in de novo RNA structure inference approaches.

Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR.

NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features. We previously defined minimal 5' leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5' leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5' leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination.

HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting.

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNALys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNALys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNALys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Unexpectedly, the affinity of MA for tRNALys3 (Kd = 0.63 ± 0.03 µM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P2-enriched liposomes (Kd(apparent) = 10.2 ± 2.1 µM), and NMR studies indicate that tRNALys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P2, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNALys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag-Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.

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 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]2; 47 nt/strand). A 9 Å 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 (10 ns) 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.