Victoria D'Souza

How retroviruses select their genomes

As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein–RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.

Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription. Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled. Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein. Here we show that dimerization-induced register shifts in base pairing within the Ψ-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 ± 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide ‘core encapsidation’ segment of the MoMuLV Ψ site reveals a network of interactions that promote sequence- and structure-specific binding by NCs single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

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).

Preformed protein-binding motifs in 7SK snRNA: structural and thermodynamic comparisons with retroviral TAR.

The 7SK small nuclear RNA is a highly conserved non-coding RNA that regulates transcriptional elongation. 7SK utilizes the HEXIM proteins to sequester the transcription factor P-TEFb by a mechanism similar to that used by retroviral TAR RNA to engage Tat and P-TEFb. Tat has also recently been shown to bind 7SK directly and recruit P-TEFb to TAR. We report here the solution structures of the free and arginine-bound forms of stem loop 4 of 7SK (7SK-SL4). Comparison of the 7SK-SL4 and TAR structures demonstrates the presence of a common arginine sandwich motif. However, arginine binding to 7SK-SL4 is mechanistically distinct and occurs via docking into a pre-organized pocket resulting in a 1000-fold increased affinity. Furthermore, whereas formation of the binding pocket in TAR requires a critical base-triple, hydrogen-bond formation between the equivalent bases in 7SK-SL4 is not essential and the pocket is stabilized solely by a pseudo base-triple platform. In addition, this theme of preformed protein binding motifs also extends into the pentaloop. The configuration of the loop suggests that 7SK-SL4 is poised to make ternary contacts with P-TEFb and HEXIM or Tat. These key differences between 7SK-SL4 and TAR present an opportunity to understand RNA structural adaptation and have implications for understanding differential interactions with Tat.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNAPro primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues—which are exactly complementary—for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNAPro reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.