Phosphorylation State Modulates Role of SARS-CoV-2 Nucleocapsid Protein in Viral Replication
Loading...
Date
2024-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
Abstract
SARS-CoV-2, the causative agent of COVID-19, is a positive-sense single-stranded betacoronavirus. Significant progress has been made in understanding the SARS-CoV-2 lifecycle. The Nucleocapsid protein (N protein) is responsible for viral RNA (vRNA) packaging, plays a vital role in replication and transcription, and is associated with viral replication-transcription complexes. N protein contains a conserved Ser-Arg (SR)-rich disordered linker between its two structured domains, which is a known site of phosphorylation by multiple host kinases; modification is proposed to regulate its function during infection. Phosphorylated N protein has been proposed to participate in viral transcription while non-phosphorylated N protein packages gRNA into new virions. However, little is known about N protein binding specificity to nucleic acids, and how it is modulated by phosphorylation. The 5' untranslated region (UTR) of vRNA has conserved secondary structures that are necessary for vRNA replication. We used RNA constructs derived from the SARS-CoV-2 5' UTR containing either stem loops (SLs) 1-4, SL5, or SL1-5, as well as HIV-1 derived RNAs. We also prepared phosphomimetic N protein mutants with 3 Ser/Thr to Asp mutations in the N-terminal region of the SR linker (3xD1) or the C-terminal region (3xD2), as well as a variant with all 6 positions mutated (6xD). Fluorescence anisotropy (FA) direct binding experiments and FA salt-titration binding assays were used to compare salt dependence and binding specificity among non-phosphorylated wild-type and mutant proteins. Trypsin time course digestion was utilized to analyze structural differences between wild-type and mutant proteins as well. Direct binding assays showed that phosphomimetic mutants and non-phosphorylated N protein have high-affinity binding (low nM Kd’s) to all RNAs tested. The non-phosphorylated N protein bound with similar specificity to all SARS2 and HIV-1 RNAs tested with a strong non-electrostatic component. The 6xD phosphomimetic N protein mutant displayed high selectivity for SARS2 RNAs and bound primarily electrostatically with HIV-1 RNAs. The location of the phosphomimetic mutations determined whether binding was primarily electrostatic (3xD1) or hydrophobic in nature (3xD2). Overall, this data supports the conclusion that the location of phosphorylation within the SR linker of SARS-CoV-2 N protein modulates its structure and binding to RNA.
Description
Keywords
SARS-CoV-2, Nucleocapsid Protein, Viral RNA, Fluorescence Anisotropy, Protein Phosphorylation, Site-directed, Ligase-Independent Mutagenesis (SLIM), Protein Characterization, Protein Purification