p72 DEAD box RNA helicase is required for optimal function of the zinc-finger antiviral protein

Abstract

The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of many viruses by preventing the accumulation of viral mRNAs in the cytoplasm. ZAP directly binds to the viral mRNAs and recruits the RNA exosome to degrade the target RNA. In the present study, we identified the p72 DEAD box RNA helicase, but not the highly similar RNA helicase p68, as a ZAP-interacting protein. The binding domain of ZAP was mapped to its N-terminal portion, whereas both the N-and C-terminal domains of p72 bound to ZAP. Overexpression of the C-terminal domain of p72 reduced ZAP's activity, whereas overexpression of the full-length p72 enhanced ZAP's activity. The RNA helicase activity was required for p72 to promote ZAP-mediated RNA degradation. Depletion of p72 by RNAi also reduced ZAP's activity but did not affect tristetraprolin-mediated RNA degradation. We conclude that p72 is required for the optimal activity of ZAP, and we propose that p72 helps to restructure the ZAP-bound target mRNA for efficient degradation.restriction ͉ RNA degredation ͉ retrovirus ͉ alphavirus T he zinc-finger antiviral protein (ZAP) was initially recovered as a host factor that inhibited the infection of cells by Moloney murine leukemia virus (MLV) (1). In addition to MLV, overexpression of ZAP also inhibits the replication of Ebola virus and Marburg virus (2), and several members of the Alphavirus genus, including Sindbis virus (SINV) (3). IFN treatment or SINV infection of murine bone marrow-derived dendritic cells significantly up-regulates the expression level of ZAP (4), suggesting that ZAP also may function as an antiviral effector in vivo. However, ZAP is not a universal antiviral factor; some viruses, including herpes simplex virus type 1 and yellow fever virus, grow normally in ZAP-expressing cells (3).ZAP inhibits virus replication by preventing the accumulation of the viral mRNA in the cytoplasm (1-3). In the N terminus of ZAP, there are four CCCH-type zinc-finger motifs (1). ZAP directly binds to specific viral mRNA sequences [ZAPresponsive element (ZRE)] through the CCCH-type zinc fingers (5). Furthermore, ZAP directly interacts with the RNAprocessing exosome (6), a 3Ј-5Ј exoribonucleases complex consisting of at least nine components (7). The current working model is that ZAP promotes the degradation of ZRE-containing mRNAs by directly binding to the target RNA and recruiting the exosome to degrade the RNA (6).The p72 RNA helicase is a member of the DEAD box family of RNA helicases, which are characterized by a conserved motif including Asp-Glu-Ala-Asp (DEAD) and involved in various biological processes (8,9). Compared with other DEAD box RNA helicases, p72 has a unique N-terminal domain containing repeats of the sequence RGG and a C-terminal domain rich in serine and glycine and terminating with a polyproline region (10). p72 is highly related to the better known p68 RNA helicase; they share Ϸ90% sequence identity in the core region spanning the conserved motifs characteristic of this family and 69.7% overall homology (10)...

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein

“…The control shRNA has been described previously (15). Sequences of the oligonucleotides are the following: TRIM25i-FP, 5=-GATCCCCGGTGGAGCA GCTACAACAATTCAAGAGATTGTTGTAGCTGCTCCACCTTTTTA-3=; TRIM25i-RP, 5=-AGCTTAAAAAGGTGGAGC AGCTACAACAATCTCTTGAATTGTTGTAGCTGCTCCACCGGG-3=.…”

“…Furthermore, ZAP recruits the deadenylase PARN to remove the poly(A) tail of target mRNA and recruits the RNA exosome, a 3=-5= exoribonuclease complex, to degrade the deadenylated RNA body from the 3= end (6,10). ZAP also recruits the decapping complex through its cofactor p72, a DEAD box RNA helicase, to remove the cap structure (6,15). The decapped RNA body is degraded by the 5=-3= exoribonuclease XrnI from the 5= end (6).…”

Glycogen Synthase Kinase 3β (GSK3β) Modulates Antiviral Activity of Zinc-finger Antiviral Protein (ZAP)

“…To generate a GSK3␤-expressing construct that cannot be down-regulated by Gi-5 shRNA, silent mutations were introduced into pCMV-HA-FLAG-GSK3␤ by overlapping PCR (8). The sequences of the primers used were as follows: GSK3␤i5M-FP, 5Ј-AAGAACCGTGAGTTGCAAAT-TATGAGAAAG-3Ј; and GSK3␤i5M-RP, 5Ј-CTTTCTCATA-ATTTGCAACTCACGGTTCTT-3Ј (with the modified nucleotides underlined).…”

“…Further studies have shown that ZAP binds directly to target viral mRNA (2), recruits the cellular polyA ribonuclease (PARN) to remove the poly(A) tail, and recruits the 3Ј-5Ј exoribonuclease complex exosome to degrade the RNA body from the 3Ј-end (2). DEAD box RNA helicase p72 is required for the optimal antiviral activity of ZAP (8). ZAP also recruits the decapping complex Dcp1-Dcp2 through p72 to remove the cap structure of the target viral mRNA to initiate the degradation of viral mRNA from the 5Ј-end (2).…”

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

“…The RNA helicase p72 is required for optimal function of ZAP (Chen et al, 2008). Insertion of some viral sequences into the 3' UTR of a luciferase reporter rendered the reporter sensitive to ZAP's inhibitory effect (Guo et al, 2004).…”