Cells have evolved multiple mechanisms to inhibit viral replication. To identify previously unknown antiviral activities, we screened mammalian complementary DNA (cDNA) libraries for genes that prevent infection by a genetically marked retrovirus. Virus-resistant cells were selected from pools of transduced clones, and an active antiviral cDNA was recovered. The gene encodes a CCCH-type zinc finger protein designated ZAP. Expression of the gene caused a profound and specific loss of viral messenger RNAs (mRNAs) from the cytoplasm without affecting the levels of nuclear mRNAs. The finding suggests the existence of a previously unknown machinery for the inhibition of virus replication, targeting a step in viral gene expression.

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3 long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.The zinc finger antiviral protein (ZAP) was originally recovered from a screen for genes conferring resistance to the infection of cells by Moloney murine leukemia virus (MLV) (11). The overexpression of ZAP rendered cells 30-fold more resistant to viral infection. An analysis to determine the step at which ZAP blocked virus infection revealed that in ZAP-expressing cells, reverse transcription and nuclear entry of the viral DNA were normal but the production of viral RNA in the cytoplasm was inhibited (11). In addition to its inhibition of MLV, ZAP potently inhibits the replication of multiple members of the Alphavirus genus of the Togaviridae family, including Sindbis virus (SIN), Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus (3). The expression of ZAP does not induce a broad-spectrum antiviral state, as some viruses, including herpes simplex virus type 1 and yellow fever virus, grow normally in ZAP-expressing cells (3). ZAP targets SIN at a stage after binding and penetration, and it prevents translation of the incoming viral RNA (3). Given that alphaviruses are replicated entirely in an RNA state in the cytoplasm (19) and that the production of MLV viral RNA was inhibited only in the cytoplasm, it is tempting to propose that a common mechanism occurring in the cytoplasm underlies the ZAPmediated elimination of MLV and SIN viral RNAs. However, since there is no obvious sequence homology between MLV and SIN, the common feature(s) shared by these two divergent viruses to account for their sensitivity to ZAP remained elusive.Sequence analysis revealed that in the N terminus of ZAP there are four CCCH-type zinc finger motifs. A fragment of 254 amino acids of the N terminus (NZAP) containing the four zinc finger motifs displa...

Zinc-finger antiviral protein (ZAP) is a host antiviral factor that specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing accumulation of the viral mRNA in the cytoplasm. In previous studies, we demonstrated that ZAP directly binds to its specific target mRNAs. In this article, we provide evidence indicating that ZAP recruits the RNA processing exosome to degrade the target RNA. ZAP comigrated with the exosome in sucrose or glycerol velocity gradient centrifugation. Immunoprecipitation of ZAP coprecipitated the exosome components. In vitro pull-down assays indicated that ZAP directly interacted with the exosome component hRrp46p and that the binding region of ZAP was mapped to amino acids 224 -254. Depletion of the exosome component hRrp41p or hRrp46p with small interfering RNA significantly reduced ZAP's destabilizing activity. These findings suggest that ZAP is a trans-acting factor that modulates mRNA stability. RNA degradationT he degradation of mRNA is an important control point in the regulation of gene expression (1-6). The general mRNA decay is initiated by removal of the poly(A) tail. The body of the RNA is degraded either from the 5Ј end by exonuclease Xrn1 after decapping or from the 3Ј end by an exoribonucleases complex named the exosome (7).RNA decay mechanisms are diverse in the extent of decay and decay characteristics. This diversity presumably arises from the diverse RNA-binding factors and the diverse RNA-protein and protein-protein interactions in the decay machinery (8-11). Many cis-acting elements and trans-acting factors have been shown to engage in mRNA turnover regulation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In mammalian cells, the most common cis element is the AU-rich element (ARE) (21-24), which has been found in the 3Ј UTR of a wide variety of short-lived mRNAs, such as those of growth factors, cytokines, and protooncogenes (25-27). Various ARE binding proteins (AUBPs) have been shown to modulate the stability of ARE-containing RNAs. Some AUBPs such as HuR (13, 28) and NF90 (29) stabilize the RNA, and other AUBPs such as tristetraprolin (TTP) (12,30,31), KRSP (32), and AUF1 (33, 34) destabilize the RNA. It has been reported that the destabilizing AUBPs recruit the exosome to degrade the ARE-containing RNAs (35).The exosome is an evolutionarily highly conserved 3Ј-5Ј exoribonucleases complex existing in both the nucleus and the cytoplasm (36-40). The nuclear exosome is required for the 3Ј processing of many RNA substrates, including prerRNA, snoRNA, snRNA, and premRNA (41-43). The nuclear exosome also functions in the surveillance system, in which the transcripts with defects generated in the RNA processing and exporting pathways are degraded (44-46). The cytoplasmic exosome plays a key role in the degradation of aberrant or unused intermediate mRNAs and AREcontaining mRNAs (1,35,(47)(48)(49)(50)(51)(52)(53). The effects on the target RNA depend on interactions among the exosome, exosome cofactors, the target RNA, and sp...