Sarah C. Keane

Structure of the HIV-1 RNA packaging signal

Structural signals that direct HIV packaging During the viral replication cycle of HIV, unspliced dimeric RNA genomes are efficiently packaged into new virions at the host cell membrane. Packaging is directed by a region at the start of the genome, the 5′ leader. The architecture of the 5′ leader remains controversial. Keane et al. developed nuclear magnetic resonance methods to determine the structure of a 155-nucleotide-long region of the 5′ leader that can direct viral packaging. The structure shows how the 5′ leader binds to the HIV protein that directs packaging, how unspliced dimeric genomes are selected for packaging, and how translation is suppressed when the genome dimerizes. Science, this issue p. 917 A nuclear magnetic resonance structure of a region of the HIV-1 RNA 5′ leader gives insight into how the viral genome is selected for packaging. The 5′ leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a 2H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; ΨCES). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

Regio-Selective Chemical-Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site‐specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically 13C‐labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost‐effective preparation of labels that are unattainable by current methods. The methodology generates versatile 13C and 15N labeling patterns which, when employed with relaxation‐optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro‐tRNA (76 nt), and HIV‐1 core encapsidation signal (155 nt).

Mouse Hepatitis Virus Stem-Loop 2 Adopts a uYNMG(U)a-Like Tetraloop Structure That Is Highly Functionally Tolerant of Base Substitutions

ABSTRACT Stem-loop 2 (SL2) of the 5′-untranslated region of the mouse hepatitis virus (MHV) contains a highly conserved pentaloop (C47-U48-U49-G50-U51) stacked on a 5-bp stem. Solution nuclear magnetic resonance experiments are consistent with a 5′-uYNMG(U)a or uCUYG(U)a tetraloop conformation characterized by an anti-C47-syn-G50 base-pairing interaction, with U51 flipped out into solution and G50 stacked on A52. Previous studies showed that U48C and U48A substitutions in MHV SL2 were lethal, while a U48G substitution was viable. Here, we characterize viruses harboring all remaining single-nucleotide substitutions in the pentaloop of MHV SL2 and also investigate the degree to which the sequence context of key pentaloop point mutations influences the MHV replication phenotype. U49 or U51 substitution mutants all are viable; C47 substitution mutants also are viable but produce slightly smaller plaques than wild-type virus. In contrast, G50A and G50C viruses are severely crippled and form much smaller plaques. Virus could not be recovered from G50U-containing mutants; rather, only true wild-type revertants or a virus, G50U/C47A, containing a second site mutation were recovered. These functional data suggest that the Watson-Crick edges of C47 and G50 (or A47 and U50 in the G50U/C47A mutant) are in close enough proximity to a hydrogen bond with U51 flipped out of the hairpin. Remarkably, increasing the helical stem stability rescues the previously lethal mutants U48C and G50U. These studies suggest that SL2 functions as an important, but rather plastic, structural element in stimulating subgenomic RNA synthesis in coronaviruses.

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

Significance A nucleotide-specific 2H-edited NMR approach was used to determine the nature of the intermolecular interface in the intact, dimeric HIV type-1 (HIV-1) 5′-leader RNA (230 kD). The studies distinguish between previously proposed extended duplex and kissing hairpin models and identify additional intermolecular interaction sites. A one-dimensional 2H-edited NMR method that allows temporal monitoring of intermolecular base-pair formation revealed that the observed “extended dimer interface” forms rapidly, even in the absence of RNA chaperones. In addition to addressing long-standing questions about retroviral genome dimerization, these studies illustrate the utility of 2H-edited NMR for determining the structures and folding kinetics of relatively large RNAs. HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

Functional transcriptional regulatory sequence (TRS) RNA binding and helix destabilizing determinants of murine hepatitis virus (MHV) nucleocapsid (N) protein.

Background: RNA binding and remodeling determinants of CoV nucleocapsid (N) proteins are poorly understood. Results: Key molecular features of RNA binding and helix destabilizing activity linked to viral replication are defined. Conclusion: Helix destabilizing activity on a short RNA duplex is strongly linked to virus replication in cultured cells. Significance: Coronaviral N protein represents an excellent target for the development of antiviral agents. Coronavirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse hepatitis virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3′-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and virus viability.

Solution Structure of Mouse Hepatitis Virus (MHV) nsp3a and Determinants of the Interaction with MHV Nucleocapsid (N) Protein

ABSTRACT Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [K a], [1.4 ± 0.3] × 106 M−1). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as K a) of 8.1 × 103 M−1. These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N.

Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another. DOI: http://dx.doi.org/10.7554/eLife.14663.001

NMR studies of the structure and function of the HIV-1 5′-leader

The 5′-leader of the human immunodeficiency virus type 1 (HIV-1) genome plays several critical roles during viral replication, including differentially establishing mRNA versus genomic RNA (gRNA) fates. As observed for proteins, the function of the RNA is tightly regulated by its structure, and a common paradigm has been that genome function is temporally modulated by structural changes in the 5′-leader. Over the past 30 years, combinations of nucleotide reactivity mapping experiments with biochemistry, mutagenesis, and phylogenetic studies have provided clues regarding the secondary structures of stretches of residues within the leader that adopt functionally discrete domains. More recently, nuclear magnetic resonance (NMR) spectroscopy approaches have been developed that enable direct detection of intra- and inter-molecular interactions within the intact leader, providing detailed insights into the structural determinants and mechanisms that regulate HIV-1 genome packaging and function.