Nucleocapsid Phosphorylation and RNA Helicase DDX1 Recruitment Enables Coronavirus Transition from Discontinuous to Continuous Transcription

Abstract

Coronaviruses contain a positive-sense singlestranded genomic (g) RNA, which encodes nonstructural proteins. Several subgenomic mRNAs (sgmRNAs) encoding structural proteins are generated by template switching from the body transcription regulatory sequence (TRS) to the leader TRS. The process preferentially generates shorter sgmRNA. Appropriate readthrough of body TRSs is required to produce longer sgmRNAs and full-length gRNA. We find that phosphorylation of the viral nucleocapsid (N) by host glycogen synthase kinase-3 (GSK-3) is required for template switching. GSK-3 inhibition selectively reduces the generation of gRNA and longer sgmRNAs, but not shorter sgmRNAs. N phosphorylation allows recruitment of the RNA helicase DDX1 to the phosphorylated-N-containing complex, which facilitates template readthrough and enables longer sgmRNA synthesis. DDX1 knockdown or loss of helicase activity markedly reduces the levels of longer sgmRNAs. Thus, coronaviruses employ a unique strategy for the transition from discontinuous to continuous transcription to ensure balanced sgmRNAs and full-length gRNA synthesis.

Cellular peptidyl-prolyl cis/trans isomerase Pin1 facilitates replication of feline coronavirus

“…We speculate that Pin1 may directly or indirectly regulate the function of GSK-3. DDX1 is a member of the DEAD-box protein family, and knockdown of DDX1 reduced the quantities of longer viral RNAs of infectious bronchitis virus (IBV) (Wu et al, 2014). DDX1 has been identified as a member of the cellular interactomes for the IBV-N protein (Emmott et al, 2013), and an interaction between DDX1 and the non-structural protein 14 (nsp14) of IBV has been identified (Xu et al, 2010).…”

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Infectious bronchitis virus (IBV) N protein was recently shown to recruit cellular helicase DDX1 to viral RTCs, facilitating TRS read-through and synthesis of long sgmRNAs (34). Interestingly, DDX1 recruitment requires N protein phosphorylation by cellular GSK3 kinase (34).…”

“…Infectious bronchitis virus (IBV) N protein was recently shown to recruit cellular helicase DDX1 to viral RTCs, facilitating TRS read-through and synthesis of long sgmRNAs (34). Interestingly, DDX1 recruitment requires N protein phosphorylation by cellular GSK3 kinase (34). Thus, the cell factor DDX1, attracted by phosphorylated N protein, provides a unique strategy for the transition from discontinuous to continuous transcription in coronaviruses to ensure balanced sgmRNA and full-length gRNA synthesis.…”

“…N protein nuclear localization is associated with induction of cell cycle arrest and inhibition of cytokinesis (68–72) and is involved in recruitment to the cytoplasm of cell nuclear proteins, such as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and the helicase DDX1 (34, 73). As noted above, N protein–recruited DDX1 functions in the RTC in facilitating TRS read-through and synthesis of long sgmRNAs (34). The 3b proteins of IBV (74) and SARS-CoV (75, 76), though different in nature, have also been located in part in the nuclei of transfected or infected cells.…”

“…SARS-CoV nsp13 has been shown to interact specifically with the cellular RNA helicase DDX5, which is involved in coronavirus RNA synthesis (141). In addition, the cellular helicase DDX1 is recruited to RTCs, and the effects of DDX1 expression knockdown indicate that it might be an essential cofactor for coronavirus RNA replication and transcription (34, 142). Interestingly, the helicase activity of nsp13 is enhanced 2-fold by nsp12 through direct protein-protein interaction (143), suggesting that interaction of these proteins in a functional RTC improves the efficiency of viral RNA synthesis.…”