SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.

Short- and long-range interactions in the HIV-1 5' UTR regulate genome dimerization and packaging.

RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.

Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.

CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting.

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.