Revolution in an RNA-packaging capsid: Artificial nucleocapsid proteins, which could be analogous to those used by viruses to package their genomes, are a promising way to protect and deliver RNAs. Using an escalating challenge by nucleases, Tetter et al. evolved a protein that forms multimeric, spherical cages into a highly efficient capsid that selectively packages its own encoding RNA. Cryo–electron microscopy of the final design and intermediates revealed a stepwise expansion in size, enabled by destabilizing amino acid substitutions and a domain swap that results in a change of oligomerization interfaces for the base units of the cage. In addition to altering the protein, directed evolution resulted in changes to the encoding RNA structure that enabled efficient uptake versus other cellular RNAs. Science , abg2822, this issue p. 1220

Significance: Single-stranded RNA viruses self-assemble protective protein containers around their cognate genomes rapidly and efficiently at low concentrations. RNA encapsidation in vivo occurs preferentially with the cognate genome, in contrast to many in vitro reassembly experiments. We describe in molecular detail how this specificity and efficiency is accomplished using multiple contacts between coat proteins and dispersed packaging signals in the viral genome. The sequences and relative positioning of the packaging signals are important for this mechanism, creating a strong evolutionary constraint. Packaging signals overlap untranslated and coding regions ensuring assembly is in competition with other functions of the genome. Disrupting these contacts has deleterious consequences for capsid assembly identifying a novel antiviral drug target.

Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein–RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.

Significance: Viruses composed of a shell of coat proteins enclosing ssRNA genomes are among the simplest biological entities. Their lifecycles include a range of processes, such as specific genome encapsidation and efficient capsid self-assembly. Until recently, these were not linked, but we have shown that many viruses in this class encode multiple, degenerate RNA sequence/structure motifs that bind cognate coat proteins collectively. This simultaneously ensures specific genome packaging and efficient virion assembly via an RNA-encoded instruction manual. Here we extract essential features of this manual in a viral RNA genome, creating a synthetic sequence with an assembly substrate superior to the natural equivalent. Such RNAs have the potential for efficient production of stable virus-like particle vaccines and/or gene/drug delivery vehicles.

Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 5′-flaps. Related to FEN specificity for substrates with free 5′-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 5′-flaps thread through the arch, which when structured can only accommodate single-stranded (ss) DNA, or the arch acts as a clamp. Here we show that free 5′-termini are selected using a disorder-thread-order mechanism. Adding short duplexes to 5′-flaps or 3′-streptavidin does not markedly impair the FEN reaction. In contrast, reactions of 5′-streptavidin substrates are drastically slowed. However, when added to premixed FEN and 5′-biotinylated substrate, streptavidin is not inhibitory and complexes persist after challenge with unlabelled competitor substrate, regardless of flap length or the presence of a short duplex. Cross-linked flap duplexes that cannot thread through the structured arch react at modestly reduced rate, ruling out mechanisms involving resolution of secondary structure. Combined results explain how FEN avoids cutting template DNA between Okazaki fragments and link local FEN folding to catalysis and specificity: the arch is disordered when flaps are threaded to confer specificity for free 5′-ends, with subsequent ordering of the arch to catalyze hydrolysis.

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.