Interactions between lysyl–tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNALys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl–tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5′UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.

The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2′-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2′-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2′-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects domestic cattle. The structural protein Gag, found in all retroviruses, is a polyprotein comprising three major functional domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Previous studies have shown that both mature BLV MA and NC are able to bind to nucleic acids; however, the viral assembly process and packaging of viral genomic RNA requires full-length Gag to produce infectious particles. Compared to lentiviruses, little is known about the structure of the Gag polyprotein of deltaretroviruses. In this work, structural models of full-length BLV Gag and Gag lacking the MA domain were generated based on previous structural data of individual domains, homology modeling, and flexible fitting to SAXS data using molecular dynamics. The models were used in molecular dynamic simulations to determine the relative mobility of the protein backbone. Functional annealing assays revealed the role of MA in the nucleic acid chaperone activity of BLV Gag. Our results show that full-length BLV Gag has an elongated rod-shaped structure that is relatively rigid, with the exception of the linker between the MA and CA domains. Deletion of the MA domain maintains the elongated structure but alters the rate of BLV Gag-facilitated annealing of two complementary nucleic acids. These data are consistent with a role for the MA domain of retroviral Gag proteins in modulating nucleic acid binding and chaperone activity. Importance BLV is a retrovirus that is found worldwide in domestic cattle. Since BLV infection has serious implications for agriculture, and given its similarities to human retroviruses such as HTLV-1, the development of an effective treatment would have numerous benefits. The Gag polyprotein exists in all retroviruses and is a key player in viral assembly. However, the full-length structure of Gag from any virus has yet to be elucidated at high resolution. This study provides structural data for BLV Gag and could be a starting point for modeling Gag-small molecule interactions with the ultimate goal of developing of a new class of pharmaceuticals. (C) 2019 Elsevier Ltd. All rights reserved.

Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly.

Human tRNALys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1) and anneals to the complementary primer binding site (PBS) in the genome. All tRNALys isoacceptors interact with human lysyl-tRNA synthetase (hLysRS) and are selectively packaged into virions. tRNALys3 must be released from hLysRS in order to anneal to the PBS, and this process is proposed to be facilitated by the interaction of hLysRS with a tRNA-like element (TLE) first identified in the HIV-1 5′-untranslated region (5′-UTR) of the subtype B NL4-3 virus. However, a significant subset of HIV-1 strains represented by the MAL isolate possess a different secondary structure in this region of the genome. Thus, to establish the conservation of this mechanism for primer targeting and release, we investigated the subtype A-like 5′-UTR of the MAL isolate. hLysRS bound to a 229-nt MAL RNA containing the PBS domain with high affinity (Kd = 47 nM), and to a 98-nt truncated construct with ∼10-fold reduced affinity. These results resemble previous studies using analogous NL4-3-derived RNAs. However, in contrast to studies with NL4-3, no binding was observed to smaller stem–loop elements within the MAL PBS domain. The tertiary structure of the 98-nt construct was analyzed using small-angle X-ray scattering, revealing remarkable global structural similarity to the corresponding NL4-3 PBS/TLE region. These results suggest that the tRNA-like structure within the 5′-UTR is conserved across distinct HIV-1 subtypes and that hLysRS recognition of the MAL isolate is likely not conferred by specific sequence elements but by 3D structure.

In HIV-1 infected cells, full-length viral genomic RNA (gRNA) is selectively packaged by the HIV-1 Gag protein despite a vast excess of spliced viral and host RNAs. The mechanism of this selective packaging is incompletely understood but a region of gRNA known as "Psi" mediates specific Gag interaction. HIV-1 Gag binds Psi and non-Psi RNA with similar affinity under physiological salt concentration (∼150 mM NaCl), but the salt dependence of these two binding events differs dramatically (Webb, JA, et al, RNA 2013). HIV-1 Gag binds Psi RNA with a strong non-electrostatic binding component and a small effective charge (Zeff ∼ 5). In contrast, HIV-1 Gag binds non-Psi RNA with a very weak non-electrostatic binding component and a larger effective charge (Zeff ∼ 9). In this work, we use salt-titration binding assays to study the effect of various Gag and Psi RNA mutations on the Psi and non-Psi binding interactions of HIV-1 and Rous sarcoma virus Gag proteins. Our findings are consistent with a model in which Gag binding to cognate Psi RNA shifts the equilibrium from a non-assembling to an assembling Gag state, providing an advantage for nucleation of assembly only on gRNA.

In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building.

HIV-1 particle assembly, which occurs at the plasma membrane (PM) of cells, is driven by the viral polyprotein Gag. Gag recognizes phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific phospholipid, via the highly basic region (HBR) in its N-terminal matrix (MA) domain. The HBR is also known to bind to RNA. We have previously shown, using an in vitro liposome binding assay, that RNA inhibits Gag binding to membranes that lack PI(4,5)P2 If this RNA block is removed by RNase treatment, Gag can bind nonspecifically to other negatively charged membranes. In an effort to identify the RNA species that confer this inhibition of Gag membrane binding, we have tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P2 We found that some tRNA species and RNAs containing stem-loop 1 of the psi region in the 5' untranslated region of the HIV-1 genome impose inhibition of Gag binding to membranes lacking PI(4,5)P2 In contrast, a specific subset of tRNAs, as well as an RNA sequence previously selected in vitro for MA binding, failed to suppress Gag-membrane interactions. Furthermore, switching the identity of charged residues in the HBR did not diminish the susceptibility of Gag-liposome binding for each of the RNAs tested, while deletion of most of the NC domain abrogates the inhibition of membrane binding mediated by the RNAs that are inhibitory to WT Gag-liposome binding. These results support a model in which NC facilitates binding of RNA to MA and thereby promotes RNA-based inhibition of Gag-membrane binding.

BACKGROUND:The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC.RESULTS:Here, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal.CONCLUSIONS:Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.

Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The “psi” (Ψ) element within the 5′-untranslated region (5′UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity.

The superfamily of 3'-5' polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNA(His) guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNA(His) maturation reaction, which is distinct from the tRNA(His) maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5'-editing in vivo and in vitro, establishing template-dependent 3'-5' polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3'-5' polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3'-5' polymerases in eukaryotes.

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.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder cause by absence of the glycosidase alpha-L-idurondase (IDUA), resulting in the systemic accumulation of glycosaminoglycan storage materials heparan sulfate and dermatan sulfate. Affected individuals exhibit a range of manifestations including hepatosplenomegaly, skeletal dysplasias, and cardiopulmonary obstruction. The severe form of the disease (Hurler syndrome) is associated with progressive neurologic dysfunction and death by age 10. Current treatments for MPS I include enzyme replacement (ERT), and hematopoietic stem cell transplantation (HSCT). However, these treatments are expensive, insufficiently effective, and allogeneic HSCT is associated with considerable morbidity and mortality. As an alternate approach to MPS I, Immusoft Corporation is developing as a cellular therapeutic primary B cells that have been genetically engineered for long-term expression and secretion of IDUA. At the core of the technology is the generation of genetically engineered autologous human B cells, which are isolated and expanded from patient blood. Mature B cells (specifically long-lived plasma cells) are the ideal cell type to produce protein since they live for long periods of time, occupy diverse tissues throughout the body and are capable of secreting very high levels of protein. In collaboration with Discovery Genomics, Inc., the University of Minnesota and the Fred Hutchinson Cancer Research Center, Immusoft has developed a treatment for Mucopolysaccharidosis type I (MPS I). In initial tests, Sleeping Beauty (SB) transposons expressing the human iduronidase gene were introduced into cultured B lymphoblastoid cells with and without a source of SB transposase, demonstrating long-term, high level expression of IDUA activity in vitro. SB transposons expressing both IDUA and a methotrexate-resistant human dihydrofolate reductase allowed for a >10-fold expansion of IDUA-expressing cells in the presence of methotrexate, as determined by intracellular staining for human IDUA and flow cytometry. Overall IDUA expression was also increased >10-fold in the methotrexate-selected cell population. IDUA encoding SB transposons were also nucleofected along with a source of SB transposase into primary human memory B cells, demonstrating expression of IDUA at levels more than 100 times greater than that observed in cultures of untreated primary B cells. These data support the development of a clinical trial to test SB-engineered B cells in the treatment of MPS I, with implications for improved treatment of other lysosomal storage disorders as well.

The field of viral molecular biology has reached a precipice for which pioneering studies on the structure of viral RNAs are beginning to bridge the gap. It has become clear that viral genomic RNAs are not simply carriers of hereditary information, but rather are active players in many critical stages during replication. Indeed, functions such as cap-independent translation initiation mechanisms are, in some cases, primarily driven by RNA structural determinants. Other stages including reverse transcription initiation in retroviruses, nuclear export and viral packaging are specifically dependent on the proper 3-dimensional folding of multiple RNA domains to recruit necessary viral and host factors required for activity. Furthermore, a large-scale conformational change within the 5'-untranslated region of HIV-1 has been proposed to regulate the temporal switch between viral protein synthesis and packaging. These RNA-dependent functions are necessary for replication of many human disease-causing viruses such as severe acute respiratory syndrome (SARS)-associated coronavirus, West Nile virus, and HIV-1. The potential for antiviral development is currently hindered by a poor understanding of RNA-driven molecular mechanisms, resulting from a lack of structural information on large RNAs and ribonucleoprotein complexes. Herein, we describe the recent progress that has been made on characterizing these large RNAs and provide brief descriptions of the techniques that will be at the forefront of future advances. Ongoing and future work will contribute to a more complete understanding of the lifecycles of retroviruses and RNA viruses and potentially lead to novel antiviral strategies.

Significance A highly conserved region of the HIV-1 RNA genome is responsible for regulating numerous steps of the retroviral life cycle, including initiation of reverse transcription. A complete understanding of the mechanisms controlling HIV-1 replication requires structural characterization of this RNA; unfortunately, however, its large size and conformational flexibility makes common methods of solving structures, such as X-ray crystallography and NMR, exceedingly difficult. The present study uses a solution technique, small-angle X-ray scattering coupled with computational molecular modeling, to characterize three ∼100-nucleotide RNAs that play central roles in HIV-1 replication. One of these domains mimics the L-shaped fold of tRNA, providing a structural basis for understanding how this genomic RNA coordinates interactions with a tRNA-binding host factor to facilitate initiation of reverse transcription. The most conserved region of the HIV type 1 (HIV-1) genome, the ∼335-nt 5′ UTR, is characterized by functional stem loop domains responsible for regulating the viral life cycle. Despite the indispensable nature of this region of the genome in HIV-1 replication, 3D structures of multihairpin domains of the 5′ UTR remain unknown. Using small-angle X-ray scattering and molecular dynamics simulations, we generated structural models of the transactivation (TAR)/polyadenylation (polyA), primer-binding site (PBS), and Psi-packaging domains. TAR and polyA form extended, coaxially stacked hairpins, consistent with their high stability and contribution to the pausing of reverse transcription. The Psi domain is extended, with each stem loop exposed for interactions with binding partners. The PBS domain adopts a bent conformation resembling the shape of a tRNA in apo and primer-annealed states. These results provide a structural basis for understanding several key molecular mechanisms underlying HIV-1 replication.