No abstract

Nucleocapsid (N) encoded by SARS-CoV-2 plays key roles in the replication cycle and is a critical serological marker. Here we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high affinity RNA binding platform. We also map the RNA binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose-coils, showing how N/RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based upon antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.

During the certification of the bulls at an artificial breeding centre for freedom from pestivirus infection, a single viraemic bull was identified, and further testing confirmed that it was persistently infected. The two-year-old bull was healthy and of similar bodyweight to its peers. Its semen was of normal quality on the basis of density, motility and morphological criteria. Approximately 600 doses of semen had been distributed for sire evaluation purposes to 97 dairy farms. An examination of the breeding records indicated a first service conception rate of 38 per cent. All but one of the 162 cows inseminated with the bull's semen were seropositive compared with 95 of 143 cows (66.4 per cent) inseminated with semen from other bulls. Virological studies of the 61 calves sired by the persistently infected bull revealed that two were persistently infected, but that the others were healthy and uninfected. It was concluded that the semen from this bull was a potential source of pestivirus infection for 'clean' herds.

When 73 heifers (60 of which were seronegative to pestivirus) were inseminated with pestivirus-contaminated semen from a transiently infected bull, the conception rate to a single insemination was found to be normal (65 per cent). Only three animals became systemically infected, as determined by viraemia and seroconversion. Pestivirus was isolated from the reproductive tracts of two of these heifers when they were slaughtered 42 or 43 days after insemination. Although the initial incidence of infection was low, a cycle of secondary transmission occurred approximately 29 days after insemination, with a further eight heifers (all seronegative) becoming infected from one group of 11 seronegative and four seropositive animals.

Protein footprinting mass spectrometry (MS), an emerging approach to elucidate higher-order structure (HOS) and binding, benefits from the iterative development of reaction strategies to expand the covalent labeling toolbox. Herein, we introduce a footprinting reagent for nucleophiles and demonstrate its efficacy for differential covalent labeling MS analysis. Benzoyl fluoride (BF), although reactive with water, is more practical for modifying nucleophilic functional groups than other acid halides and serves as an acyl-transfer reagent for proteins. BF is 10 times more reactive with phenolic Tyr than the current generation nucleophile footprinter. BF modifies, in addition to Tyr, Lys, His, and the N-terminus, weak nucleophiles Ser and Thr, for which few footprinters exist, imparting broad applicability with a range of nucleophiles. We applied benzoylation to a model Ser- and Thr-rich protein–ligand binding system without perturbing the protein HOS. This efficacious footprinting method expands the toolbox of reagents and provides promise for future reaction strategies including possibly membrane proteins.