Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways. Fibroblast growth factor (FGF)1 was identified as an activity that stimulates the proliferation of NIH3T3 cells (1). Currently, FGFs comprise a family of nine structurally related proteins (FGF 1-9). FGFs are expressed in specific spatial and temporal patterns and are involved in developmental processes, angiogenesis, wound healing, and tumorigenesis (2-5).FGFs bind and activate high-affinity receptor tyrosine kinases. The cloning of FGF receptors (FGFRs) has identified four distinct genes (6 -13). These receptors bind members of the FGF family with varying affinity (13-16), and alternative mRNA splicing leads to isoforms of these receptors which have unique ligand binding properties (15,17,18). An additional mechanism regulating FGF activity involves heparin or heparan sulfate proteoglycans, molecules which facilitate ligandreceptor interactions (12,19,20). FGFRs contain an extracellular ligand binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain. The extracellular domain determines ligand binding specificity and mediates ligand-induced receptor dimerization. Dimerization in turn results in one or more trans-phosphorylation events and the subsequent activation of the receptor (21).The extracellular region of the FGFR contains three immunoglobulin-like (Ig-like) domains (6). Alternative mRNA splicing creates several forms of the FGF receptor which differ in their extracellular sequence and have unique ligand binding properties. One splicing event results in the skipping of exons encoding the amino-terminal Ig-like domain (domain I) resulting in a "short" two Ig-like domain form of the receptor (22). The ligand binding properties of the short (two Ig-like domain) and long (three Ig-like domain) FGFRs are similar.2 However, the short form of the receptor may have a higher affinity for some FGFs than the long form (23). Changes in this alternative splicing pattern may correlate with the progression of se...

The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (Mpros), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV Mpros, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

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 rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.A previously unknown rat protein, designated zinc-finger antiviral protein (ZAP), was recently found to exhibit antiviral activity against Moloney murine leukemia virus (MMLV), a member of the Retroviridae. When challenged with an ecotropic MMLV carrying a luciferase reporter, cells expressing ZAP expressed 30 times less luciferase than did control cells. Expression of either the full-length rat ZAP or the aminoterminal one-third fused to the product of the zeocin resistance gene (NZAP-Zeo) was inhibitory, and the mechanism of the block was found to be a dramatic and specific loss of viral mRNAs from the cytoplasm, but not the nuclei, of cells (5). In our effort to better understand virus-host interactions, we tested ZAP's ability to inhibit infection by other viruses. Our studies indicate that, in addition to inhibiting MMLV replication, ZAP's range of targets also includes multiple members of the Alphavirus genus of the Togaviridae.Alphaviruses cause significant morbidity and mortality worldwide (reviewed in reference 7). The broad host range for these viruses includes vertebrates and invertebrates, with arthropods being the usual vectors of transmission to mammals. Infection with Sindbis virus (SIN), the type alphavirus, can lead to a painful polyarthritis, while disease caused by Venezuelan equine encephalitis virus (VEE) ranges from a mild influenzatype illness to fatal encephalitis. Alphaviruses are small, enveloped RNA viruses with an icosahedral nucleocapsid (reviewed in reference 22). The SIN genome consists of a single, capped, positive-sense RNA molecule of approximately 11.7 kb and contains a 5Ј untranslated region (UTR) as well as a 3Ј UTR and a poly(A) tail. The 5Ј-terminal two-thirds of the genomic 49S RNA is directly translated to produce the four nonstructural proteins (nsPs), while the structural proteins are encoded by a subgenomic 26S ...

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...

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

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...

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.