Guangxia Gao

Expression of the Zinc-Finger Antiviral Protein Inhibits Alphavirus Replication

ABSTRACT The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAPs 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.

The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA.

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 ZAPs destabilizing activity. These findings suggest that ZAP is a trans-acting factor that modulates mRNA stability.

The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs

ABSTRACT 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 ZAPs activity, whereas disruption of the first and third fingers just slightly lowered its activity.

Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

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 3′ end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5′ end. Depletion of each of these mRNA degradation enzymes reduced ZAPs activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5′ and 3′ mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.

Heparin can activate a receptor tyrosine kinase.

Heparin, a densely sulfated glycosaminoglycan produced by mast cells, is best known for its inhibitory effects on the blood coagulation system. Heparin or heparan sulfate proteoglycans are also essential cofactors for the interaction of fibroblast growth factors (FGFs) with their receptor tyrosine kinases (FGFRs). Here we show that heparin is a growth factor‐independent activating ligand for FGFR‐4. Heparin stimulates FGFR‐4 autophosphorylation on transfected myoblasts, fibroblasts and lymphoid cells, and is most potent on cells lacking surface heparan proteoglycan. Two functional analogs of heparin, fucoidan and dextran sulfate, are also activators of FGFR‐4, while neither heparin nor its analogs can stimulate FGFR‐1 in the absence of FGF. A mutation in the FGFR‐4 ectodomain which impairs receptor activation by FGFs does not interfere with activation by heparin, demonstrating that receptor domains required for heparin or FGF activation are not identical. Heparin activation of FGFR‐4 or of a chimeric receptor bearing FGFR‐4 ectodomain and FGFR‐1 cytodomain triggers downstream tyrosine phosphorylation of several signaling proteins, and induces proliferation of cells bearing the chimeric receptor. Consistent with these findings, a soluble FGFR‐4 ectodomain has strong FGF‐independent affinity for immobilized heparin resin, while soluble FGFR‐1 requires FGF for stable heparin interaction. Heparin activation of FGFR‐4 is the first example of a mammalian polysaccharide serving as a signaling ligand.

LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

Liver cancer stem cells (CSCs) may contribute to the high rate of recurrence and heterogeneity of hepatocellular carcinoma (HCC). However, the biology of hepatic CSCs remains largely undefined. Through analysis of transcriptome microarray data, we identify a long noncoding RNA (lncRNA) called lncBRM, which is highly expressed in liver CSCs and HCC tumours. LncBRM is required for the self-renewal maintenance of liver CSCs and tumour initiation. In liver CSCs, lncBRM associates with BRM to initiate the BRG1/BRM switch and the BRG1-embedded BAF complex triggers activation of YAP1 signalling. Moreover, expression levels of lncBRM together with YAP1 signalling targets are positively correlated with tumour severity of HCC patients. Therefore, lncBRM and YAP1 signalling may serve as biomarkers for diagnosis and potential drug targets for HCC.

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

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 ZAPs activity, whereas overexpression of the full-length p72 enhanced ZAPs activity. The RNA helicase activity was required for p72 to promote ZAP-mediated RNA degradation. Depletion of p72 by RNAi also reduced ZAPs 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.

ZAP-mediated mRNA degradation

The zinc-finger antiviral protein (ZAP) is a host factor that inhibits the replication of many viruses by preventing the accumulation of viral mRNAs in the cytoplasm. ZAP specifically binds to the viral mRNA and recruits the cellular RNA degradation machinery to degrade the target RNA. In this article, we will review the work to date in understanding the mechanisms by which ZAP promotes viral mRNA decay and discuss future research directions to further investigate the function and underlying mechanisms of ZAP.

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cells endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production.

Structure of N-terminal domain of ZAP indicates how a zinc-finger protein recognizes complex RNA

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.