The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication

Abstract

The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.Coronaviruses cause severe diseases in humans and many other animal species. Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of SARS (34, 45). Viruses in this family contain a single-stranded, positive-sense RNA genome of 27 to 31 kb. In cells infected with coronaviruses, six to nine mRNA species, including the genome-length mRNA1 and five to eight subgenomic mRNAs (mRNAs 2 to 9), are produced by a discontinuous RNA transcription mechanism (40,46,47). Avian infectious bronchitis virus (IBV), a prototype group 3 coronavirus, causes an acute and highly contagious disease in chickens, with a significant impact on the poultry industry worldwide. In IBV-infected cells, six mRNA species are produced (5). Subgenomic mRNAs 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The 5Ј two-thirds of mRNA1 comprises two large open reading frames (ORFs), 1a and 1b, and encodes polyproteins 1a and 1ab. The two polyproteins are proteolytically cleaved by virus-encoded proteinases into 15 functional nonstructural proteins (nsp2 to nsp16) (18, 25-27, 30-32, 37-39, 54, 59).The functional roles of coronavirus nonstructural proteins in replication and transcription of viral RNAs are beginning to emerge. For example, nsp14, nsp15, and nsp16 are predicted to possess exonuclease (ExoN), uridylate-specific endoribonuclease...

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…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.…”

KSHV Rta Promoter Specification and Viral Reactivation

“…K-RBP binds to DNAs that contain a GC-rich core, including a 40-bp region of the Mta promoter that contains the RBP-Jk site and overlaps CANT repeats. K-RBP competes with K-Rta to bind the Mta promoter, and represses KSHV lytic reactivation (Yang and Wood, 2007; Yang et al, 2009). …”

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

“…DDX3 inhibits hepatitis B virus reverse transcription by incorporation into nucleocapsids (Wang et al, 2009b). DDX1 can also stimulate the replication of IBV (Xu et al, 2010). It has been reported that DDX6, DDX28, DDX42, DHX15 and DDX56 are important for infectivity of West Nile virus (Chahar et al, 2013;Krishnan et al, 2008;Xu et al, 2011;Xu and Hobman, 2012).…”