The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m⁷G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.     

A possible complex containing RNA processing enzymes

The three enzymes, RNAase III, RNAase E and RNAase P participate in the processing of RNA precursors in $\text{Escherichia coli}$. In extracts which contain a mutated RNAase III or RNAase E under certain conditions RNAase P activity is not expressed while in the wild-type extract it is. Upon high-speed centrifugation of a cell extract from a strain of $\text{E.}$,$\text{coli}$, which contains all these three enzymes, the majority of RNAase P, RNAase III and RNAase E activities sediment as particles heavier than their known sizes. In a sucrose density gradient of the cell extract, part of RNAase E and RNAase P activities co-sediment while most of the RNAase III activity is found toward the top of the gradient. This behavior is distinct from other ribonucleases such as RNAase II and RNAase H, which do not sediment as complexes. This complex does not seem to be caused merely by the association of the enzymes with ribosomes.     

The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes

The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivo interactions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding of Schizosaccharomyces pombe guanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO4, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD.     

Structure of a hexameric RNA packaging motor in a viral polymerase complex

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.     

RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

A multiprotein complex that interacts with RNA polymerase II elongator

A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.     

Allosteric transcription stimulation by RNA polymerase II super elongation complex

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Rapid identification of DNA fragments containing promoters for RNA polymerase II

We describe a direct procedure for screening genomic recombinant DNA libraries or restriction fragments of cloned DNA regions for RNA polymerase II promoters. Cellular polyadenylated mRNA is chemically de-capped by beta-elimination reaction and enzymatically re-capped with [alpha-32P]GTP by vaccinia guanylyl transferase. Since this enzyme only accepts di- or triphosphorylated 5' termini as a substrate, the mRNAs are labeled exclusively at the first nucleotide, irrespective of whether the mRNA was intact or fragmented before in vitro capping. By using in vitro-capped mRNA as a hybridization probe, recombinant DNA molecules or restriction fragments that carry a cap site (and thus likely an RNA polymerase II promoter) can directly be identified. Here, we demonstrate the applicability of this procedure by the isolation and characterization of several genomic DNA clones containing RNA polymerase II promoter sequences, that are highly active in liver.