The Coronavirus helicase in replication.

The coronavirus nonstructural protein (nsp) 13 encodes an RNA helicase (nsp13-HEL) with multiple enzymatic functions, including unwinding and nucleoside phosphatase (NTPase) activities. Attempts for enzymatic inactivation have defined the nsp13-HEL as a critical enzyme for viral replication and a high-priority target for antiviral development. Helicases have been shown to play numerous roles beyond their canonical ATPase and unwinding activities, though these functions are just beginning to be explored in coronavirus biology. Recent genetic and biochemical studies, as well as work in structurally-related helicases, have provided evidence that supports new hypotheses for the helicase's potential role in coronavirus replication. Here, we review several aspects of the coronavirus nsp13-HEL, including its reported and proposed functions in viral replication and highlight fundamental areas of research that may aid the development of helicase inhibitors.

Impact of early enteral feed composition on the rate of chyle leak post-esophagectomy.

Patients undergoing esophagectomy are at risk of malnutrition and benefit from perioperative enteral feeding. Esophagectomy carries a risk of chyle leak, and this risk may be influenced by early enteral feed composition. We evaluated the impact of early enteral medium-chain triglyceride-rich feed on the prevalence and severity of chyle leak post-esophagectomy, length of stay, and postoperative weight change. This retrospective study included consecutive patients undergoing esophagectomy at a single center between January 2015 and December 2022. Patients received enteral feed on postoperative days 1-5 with Nutrison Energy or Protein Plus Energy ('standard') (January 2015- June 2021) or Nutrison Peptisorb Plus High Energy High Protein ('HEHP') enteral feed (June 2021 to December 2022). All patients transitioned to 'standard' supplemental jejunal feeding on postoperative day 6 onwards and were discharged on oral IDDSI level 4 diet. Patients who did not commence early enteral feeding were excluded from analysis. A total of 329 patients were included. Patients who received early HEHP feed had fewer chyle leaks (5/52; 9.6%) compared with patients who received standard feed (68/277; 24.5%, P = 0.017). The HEHP group had a shorter total length of hospital stay (P = 0.011). Weight change from preoperative baseline was equivalent in both groups at 6 weeks (P = 0.066) and 3 months (P = 0.400). In the context of routine jejunostomy use and early enteral feeding post-esophagectomy, HEHP feed on postoperative days 1-5 was associated with significantly fewer chyle leaks and shorter length of stay compared with standard feed. No difference was noted in postoperative weight change between groups.

A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance.

Coronaviruses (CoVs) encode nonstructural proteins 1-16 (nsps 1-16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7-16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.