Amy M. Barker

Packaging signals in single-stranded RNA viruses: nature’s alternative to a purely electrostatic assembly mechanism

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA–coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.

Summary Simple, spherical RNA viruses have well-understood, symmetric protein capsids, but little structural information is available for their asymmetric components, such as minor proteins and their genomes, which are vital for infection. Here, we report an asymmetric structure of bacteriophage MS2, attached to its receptor, the F-pilus. Cryo-electron tomography and subtomographic averaging of such complexes result in a structure containing clear density for the packaged genome, implying that the conformation of the genome is the same in each virus particle. The data also suggest that the single-copy viral maturation protein breaks the symmetry of the capsid, occupying a position that would be filled by a coat protein dimer in an icosahedral shell. This capsomere can thus fulfill its known biological roles in receptor and genome binding and suggests an exit route for the genome during infection.

Structure‐guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release

Current interferon‐based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct‐acting antivirals (DAA) with the first protease‐targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)‐based methods. This represents atomic resolution information for a full‐length virus‐coded ion channel, or “viroporin,” whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug‐protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000‐fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. Conclusion: This proof‐of‐principle that structure‐guided design can lead to drug‐like molecules affirms p7 as a much‐needed new target in the burgeoning era of HCV DAA. (Hepatology 2014;59:408–422)

Sequence-Specific, RNA-Protein Interactions Overcome Electrostatic Barriers Preventing Assembly of Satellite Tobacco Necrosis Virus Coat Protein

We have examined the roles of RNA–coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises a β-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem–loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T = 1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3 Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8–11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal–CP interactions.

Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus

We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.

Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid

ABSTRACT Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.

Distinguishing closely-related amyloid precursors using an RNA aptamer

Background: Altering the co-polymerization of proteins into amyloid fibrils provides an opportunity for manipulating fibril assembly. Results: NMR and kinetic analysis showed that an RNA aptamer distinguishes between two highly similar co-aggregating proteins. Conclusion: RNA aptamers are specific and discriminatory probes able to modulate amyloid formation. Significance: Aptamers can be used as tools to differentiate amyloid precursors that are closely related and alter assembly. Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β2-microglobulin (hβ2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ2m with an EC50 of ∼200 nm. NMR spectroscopy was used to assign the 1H-15N HSQC spectrum of the B6-hβ2m complex, revealing that the aptamer binds to the face of hβ2m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.

Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction‐modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res 37, 4887–4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site‐directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction.