The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 39 end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 59 end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 59 and 39 mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.retrovirus | host restriction factor | RNA degradation

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Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, such as HIV-1, by targeting viral mRNA for degradation. How ZAP recognizes its target RNA has been unclear. Here we report the crystal structure of the N-terminal domain of rat ZAP (NZAP225), the major functional domain. The overall structure of NZAP225 resembles a tractor, with four zinc-finger motifs located at the bottom. Structural and functional analyses identified multiple positively charged residues and two putative RNA-binding cavities forming a large putative RNA-binding cleft. ZAP molecules interact to form a dimer that binds to a ZAP-responsive RNA molecule containing two ZAP-binding modules. These results provide insights into how ZAP binds specifically to complex target RNA.

MCP-1-induced protein 1 (MCPIP1) plays an important role in the downregulation of the LPS-induced immune response by acting as an RNase targeting IL-6 and IL-12b mRNAs. A conserved domain located in the N-terminal part of MCPIP1 is thought to be responsible for its RNase activity, but its catalytic mechanism is not well understood due to the lack of an atomic resolution structure. We determined the 3D crystal structure of this MCPIP1 N-terminal conserved RNase domain at a resolution of 2.0 Å. The overall structure of MCPIP1 N-terminal conserved domain shares high structural homology with PilT N-terminal domain. We show that the RNase catalytic center is composed of several acidic residues, verifying their importance by site-specific mutagenesis. A positively charged arm close to the catalytic center may act as an RNA substrate-binding site, since exchange of critical positively charged residues on this arm with alanine partially abolish the RNase activity of MCPIP1 in vivo. Our structure of the MCPIP1 N-terminal conserved domain reveals the details of the catalytic center and provides a greater understanding of the RNA degradation mechanism.

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)...

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