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...

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BackgroundA lot of microRNAs (miRNAs) derived from viral genomes have been identified. Many of them play various important roles in virus replication and virus-host interaction. Cellular miRNAs have been shown to participate in the regulation of HIV-1 viral replication, while the role of viral-encoded miRNAs in this process is largely unknown.ResultsIn this report, through a strategy combining computational prediction and deep sequencing, we identified a novel HIV-1-encoded miRNA, miR-H3. MiR-H3 locates in the mRNA region encoding the active center of reverse transcriptase (RT) and exhibits high sequence conservation among different subtypes of HIV-1 viruses. Overexpression of miR-H3 increases viral production and the mutations in miR-H3 sequence significantly impair the viral replication of wildtype HIV-1 viruses, suggesting that it is a replication-enhancing miRNA. MiR-H3 upregulates HIV-1 RNA transcription and protein expression. A serial deletion assay suggests that miR-H3 targets HIV-1 5′ LTR and upregulates the promoter activity. It interacts with the TATA box in HIV-1 5′ LTR and sequence-specifically activates the viral transcription. In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).ConclusionsWe have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.

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)...

Three types of editosomes, each with an identical core containing six related KREPA proteins, catalyze the U insertion and deletion RNA editing of mitochondrial mRNAs in trypanosomes. Repression of expression of one of these, KREPA3 (also known as TbMP42), shows that it is essential for growth and in vivo editing in both procyclic (PF) and bloodstream (BF) life cycle stages of Trypanosoma brucei. RNA interference knockdown results in editosome disruption and altered in vitro editing in PFs, while repression by regulatable double knockout results in almost complete loss of editosomes in BFs. Mutational analysis shows that the KREPA3 zinc fingers and OB-fold domain are each essential for growth and in vivo editing. Nevertheless, KREPA3 with mutated zinc fingers incorporates into editosomes that catalyze in vitro editing and thus is not essential for editosome integrity, although stability is affected. In contrast, the OB-fold domain is essential for editosome integrity. Overall, KREPA3, especially its OB-fold, functions in editosome integrity, and its zinc fingers are essential for editing in vivo but not for the central catalytic steps. KREPA3 may function in editosome organization and/or RNA positioning.RNA editing in trypanosomes creates mature mitochondrial (mt) mRNAs by the insertion and deletion of uridylates (U's) as specified by the sequences of small guide RNAs (gRNAs) (6, 52). It employs several multiprotein complexes, one of which is the editosome that sediments at ϳ20S in glycerol gradients and contains the enzymes that catalyze the key catalytic steps of pre-mRNA cleavage, U insertion or deletion, and RNA ligation (32,50,53). The pre-mRNA becomes partially duplexed with gRNA 3Ј to the editing site (ES), after which editing occurs by cycles of enzymatic steps. The premRNA is cleaved at the ES by the KREN1 endonuclease that is specific for deletion sites or by KREN2 or KREN3, which are specific for different insertion sites. One or more U's are added to the 5Ј mRNA fragment by the KRET2 3Ј terminal uridylyl transferase or U's are removed by the KREX1 or KREX2 3Ј exonuclease, with the number of added or removed U's specified by the gRNA sequence. The processed 5Ј fragment is then religated with the 3Ј fragment by the KREL1 or KREL2 RNA ligase (3, 10-12, 15, 16, 27, 45, 48, 56). Multiple cycles of editing extend the amount of pre-mRNA/gRNA duplex, generally in the 5Ј direction, until they are completely complementary. The editing of most mRNAs requires multiple gRNAs with those specifying editing of the more 3Ј regions creating a pre-mRNA sequence that is complementary to the 5Ј "anchor" sequence of the subsequent gRNA thus providing for their orderly use. Other complexes are involved in editing, including the MRP1/MRP2, RBP16, TbRGG1, and RET1 complexes, which appear to preprocess and traffic the mRNAs and gRNAs and regulate editing (2,33,34,42,57). Other proteins that are not associated with these complexes and have not yet been identified may function as accessory factors.There are three types of edito...