The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication.

Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes. IMPORTANCE Replicating large genomes represents a challenge for RNA viruses because fast RNA synthesis is needed to escape innate immunity defenses, but faster polymerases are inherently low-fidelity enzymes. Nonetheless, the coronaviruses replicate their ≈30-kb genomes using the core polymerase structure and mechanism common to all positive-strand RNA viruses. The classic explanation for their success is that the large-genome nidoviruses have acquired an exonuclease-based repair system that compensates for the high polymerase mutation rate. In this work, we establish that the nidoviral polymerases themselves also play a key role in maintaining genome integrity via mutations at two key active-site residues that enable very fast replication rates while maintaining typical mutation rates. Our findings further demonstrate the evolutionary plasticity of the core polymerase platform by showing how it has adapted during the expansion from short-genome picornaviruses to long-genome nidoviruses.

Occurrence, function, and biosynthesis of mycofactocin.

Mycofactocin is a member of the rapidly growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Although the mycofactocin biosynthetic pathway is widely distributed among Mycobacterial species, the structure, function, and biosynthesis of the pathway product remain unknown. This mini-review will discuss the current state of knowledge regarding the mycofactocin biosynthetic pathway. In particular, we focus on the architecture and distribution of the mycofactocin biosynthetic cluster, mftABCDEF, among the Actinobacteria phylum. We discuss the potential molecular and physiological role of mycofactocin. We review known biosynthetic steps involving MftA, MftB, MftC, and MftE and relate them to pyrroloquinoline quinone biosynthesis. Lastly, we propose the function of the remaining putative biosynthetic enzymes, MftD and MftF.