The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Katalin A. Hudak - One of the best experts on this subject based on the ideXlab platform.
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pokeweed Antiviral Protein restores levels of cellular apobec3g during hiv 1 infection by depurinating vif mrna
Antiviral Research, 2015Co-Authors: Gabriela Krivdova, Katalin A. HudakAbstract:Pokeweed Antiviral Protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral Proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoProtein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its Antiviral activity.
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homodimerization of pokeweed Antiviral Protein as a mechanism to limit depurination of pokeweed ribosomes
Molecular Plant Pathology, 2010Co-Authors: Marina E Tourlakis, Rajita A Karran, Leroi V Desouza, Katalin A. HudakAbstract:SUMMARY Ribosome inactivating Proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit Protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed Antiviral Protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the Protein that escape transport to the cell wall by the endomembrane system.
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pokeweed Antiviral Protein depurinates the sarcin ricin loop of the rrna prior to binding of aminoacyl trna to the ribosomal a site
RNA, 2006Co-Authors: Sheila Mansouri, Emad Nourollahzadeh, Katalin A. HudakAbstract:Ribosome-inactivating Proteins, such as the pokeweed Antiviral Protein (PAP), inhibit translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited. Though the consequences of depurination are well characterized, the ribosome conformation required for depurination to take place has not been described. In this report, we correlate biochemical and genetic data to conclude that pokeweed Antiviral Protein depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied. We show that prior incubation of ribosomes with puromycin, an analog of the 3′-terminus of aminoacyl-tRNA, inhibits both binding and depurination by PAP in a concentration-dependent manner. Expression of PAP in the yeast strain mak8-1 results in little depurination unless the cells are lysed, a process that would promote loss of aminoacyl-tRNA from the ribosome. The mak8-1 strain is known to exhibit a higher affinity for aminoacyl-tRNA compared with wild-type cells, and therefore, its ribosomes are more resistant to PAP in vivo. These data contribute to the mechanism of action of pokeweed Antiviral Protein; specifically, they have uncovered the ribosomal conformation required for depurination that leads to subsequent translation inhibition.
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pokeweed Antiviral Protein inhibits brome mosaic virus replication in plant cells
Journal of Biological Chemistry, 2005Co-Authors: Daniel Picard, Cheng C Kao, Katalin A. HudakAbstract:Pokeweed Antiviral Protein (PAP) is a ribosome-inactivating Protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral Protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the Antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.
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generation of pokeweed Antiviral Protein mutations in saccharomyces cerevisiae evidence that ribosome depurination is not sufficient for cytotoxicity
Nucleic Acids Research, 2004Co-Authors: Katalin A. Hudak, Bijal A Parikh, Rong Di, Marianne Baricevic, Mirjana Seskar, Maria Santana, Nilgun E. TumerAbstract:Pokeweed Antiviral Protein (PAP) is a ribosome-inactivating Protein that depurinates the highly conserved α-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5′ and the 3′ ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.
Margaret R Macdonald - One of the best experts on this subject based on the ideXlab platform.
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structure of the zinc finger Antiviral Protein in complex with rna reveals a mechanism for selective targeting of cg rich viral sequences
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jennifer L Meagher, Margaret R Macdonald, Matthew A Takata, Daniel Goncalvescarneiro, Sarah C Keane, Antoine Rebendenne, Heley Ong, Victoria K Orr, Jeanne A Stuckey, Paul D. BieniaszAbstract:Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger Antiviral Protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a Protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective Antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.
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characterization of novel splice variants of zinc finger Antiviral Protein zap
Journal of Virology, 2019Co-Authors: Eduardo G Aguilar, Charles M. Rice, William M Schneider, Henrik Molina, Eleftherios Michailidis, Jonathan Pabon, Paul Park, Ype P De Jong, Margaret R MacdonaldAbstract:Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad Antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor: zinc finger Antiviral Protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C termini: ZAPL (long) encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously, it has been shown that ZAPL is more Antiviral than ZAPS, while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP: ZAPXL (extralong) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct Antiviral and/or cellular functions. By taking a gene-knockout-and-reconstitution approach, we have characterized the Antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad-spectrum Antiviral factor.IMPORTANCE ZAP is an IFN-induced host factor that can inhibit a wide range of viruses, and there is great interest in fully characterizing its Antiviral mechanism. This is the first study that defines the Antiviral capacities of individual ZAP isoforms in the absence of endogenous ZAP expression and, hence, cross talk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms: ZAPS, ZAPM, ZAPL, and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV, while all isoforms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the abilities of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.
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sindbis virus can exploit a host Antiviral Protein to evade immune surveillance
Journal of Virology, 2016Co-Authors: Xinlu Wang, Charles M. Rice, Margaret R Macdonald, Jing Zhao, Xiang Gao, Guangxia GaoAbstract:Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of Antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger Antiviral Protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs, repressing the translation of target mRNA, and promoting the degradation of target mRNA. We report here that murine embryonic fibroblast cells from ZAP knockout mice supported more efficient SVNI replication than wild-type cells. SVNI infection of 10-day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23-day-old weanling mice, whose immune system is more developed than that of the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor-neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wild-type mice. These results uncover a mechanism by which SVNI exploits a host Antiviral factor to evade innate immune surveillance. Importance Sindbis virus, a prototypic member of the Alphavirus genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host Antiviral factor that potently inhibits Sindbis virus replication in cell culture. We show here that infection of ZAP knockout suckling mice with an SVNI led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host Antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.
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multiple interferon stimulated genes synergize with the zinc finger Antiviral Protein to mediate anti alphavirus activity
PLOS ONE, 2012Co-Authors: Sophiya Karki, Charles M. Rice, John W Schoggins, Suyan Tian, Margaret R MacdonaldAbstract:The zinc finger Antiviral Protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.
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the zinc finger Antiviral Protein acts synergistically with an interferon induced factor for maximal activity against alphaviruses
Journal of Virology, 2007Co-Authors: Margaret R Macdonald, Erica S Machlin, Owen R Albin, David E LevyAbstract:Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular Antiviral state. Overexpression of the zinc finger Antiviral Protein (ZAP) imparts a cellular Antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN′s enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited Antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent Antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block Protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal Antiviral activity and that ZAP's intrinsic Antiviral activity on virion production and cell survival can have cell-type-specific outcomes.
Nilgun E. Tumer - One of the best experts on this subject based on the ideXlab platform.
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characterization of pokeweed Antiviral Protein binding to mrna cap analogs competition with nucleotides and enhancement by translation initiation factor iso4g
Biochimica et Biophysica Acta, 2009Co-Authors: Amy E Baldwin, Nilgun E. Tumer, Dixie J Goss, Mateen A Khan, Diana E FriedlandAbstract:Abstract Pokeweed Antiviral Protein (PAP) is a type I ribosomal inactivating Protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of Protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m 7 GTP. We report a binding affinity of 43.3 ± 0.1 nM at 25 °C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding Proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m 7 GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with T Δ S ° contributing 15% to the overall value of Δ G °. This is in contrast to the wheat cap-binding Proteins which are enthalpically driven in the Δ G ° for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a Protein–Protein interaction with a K d of 108.4 ± 0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m 7 GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.
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the c terminus of pokeweed Antiviral Protein has distinct roles in transport to the cytosol ribosome depurination and cytotoxicity
Plant Journal, 2007Co-Authors: Ulku Baykal, Nilgun E. TumerAbstract:Summary Pokeweed Antiviral Protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating Protein (RIP) that depurinates ribosomes at the α-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature Protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.
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evidence for retro translocation of pokeweed Antiviral Protein from endoplasmic reticulum into cytosol and separation of its activity on ribosomes from its activity on capped rna
Biochemistry, 2005Co-Authors: Bijal A Parikh, Ulku Baykal, Nilgun E. TumerAbstract:Pokeweed Antiviral Protein (PAP) is a single-chain ribosome inactivating Protein (RIP) that binds to ribosomes and depurinates the highly conserved α-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. Here, we show that both precursor and mature forms of PAP are localized in the endoplasmic reticulum (ER) in yeast. The mature form is retro-translocated from the ER into the cytosol where it escapes degradation unlike the other substrates of the retro-translocation pathway. A mutation of a highly conserved asparagine residue at position 70 (N70A) delays ribosome depurination and the onset of translation arrest. The ribosomes are eventually depurinated, yet cytotoxicity and loss of viability are markedly absent. Analysis of the variant Protein, N70A, does not reveal any decrease in the rate of synthesis, subcellular localization, or the rate of transport into the cytosol. N70A destabilizes its own...
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generation of pokeweed Antiviral Protein mutations in saccharomyces cerevisiae evidence that ribosome depurination is not sufficient for cytotoxicity
Nucleic Acids Research, 2004Co-Authors: Katalin A. Hudak, Bijal A Parikh, Rong Di, Marianne Baricevic, Mirjana Seskar, Maria Santana, Nilgun E. TumerAbstract:Pokeweed Antiviral Protein (PAP) is a ribosome-inactivating Protein that depurinates the highly conserved α-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5′ and the 3′ ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.
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pokeweed Antiviral Protein regulates the stability of its own mrna by a mechanism that requires depurination but can be separated from depurination of the α sarcin ricin loop of rrna
Journal of Biological Chemistry, 2002Co-Authors: Bijal A Parikh, Chris Coetzer, Nilgun E. TumerAbstract:Abstract Pokeweed Antiviral Protein (PAP), a single chain ribosome-inactivating Protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, α-sarcin/ricin loop in the large rRNA, resulting in inhibition of Protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeastSaccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to theN-glycosidase activity of the Protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.
Stefan Rothenburg - One of the best experts on this subject based on the ideXlab platform.
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orthopoxvirus k3 orthologs show virus and host specific inhibition of the Antiviral Protein kinase pkr
PLOS Pathogens, 2021Co-Authors: Chorong Park, Greg Brennan, Chen Peng, Julhasur M Rahman, Sherry L Haller, Loubna Tazi, Stefan RothenburgAbstract:The Antiviral Protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host Proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.
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orthopoxvirus k3 orthologs show virus and host specific inhibition of the Antiviral Protein kinase pkr
bioRxiv, 2020Co-Authors: Chorong Park, Greg Brennan, Chen Peng, Julhasur M Rahman, Sherry L Haller, Loubna Tazi, Stefan RothenburgAbstract:The Antiviral Protein kinase R (PKR) is an important restriction factor, which poxviruses must overcome to productively infect host cells. Many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus, to inhibit PKR. Although the interaction between PKR and eIF2alpha is highly conserved, some K3 orthologs were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that the amino acid combinations needed to mediate improved or diminished inhibition of PKR varied between different species. The data presented here show that even closely related species can display drastically different sensitivities to orthopoxvirus PKR inhibitors, and that orthologs from closely related poxviruses can show strong differences in their function. Furthermore, there is likely to be a limited number of possible mutations that disrupt K3-interactions but still maintain PKR/eIF2alpha interactions. Thus, by chance some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host Proteins to virus inhibition nor the effectiveness of viral host antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.
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Species-specific inhibition of Antiviral Protein kinase R by capripoxviruses and vaccinia virus.
Annals of the New York Academy of Sciences, 2019Co-Authors: Chorong Park, Greg Brennan, Chen Peng, Stefan RothenburgAbstract:Double-stranded RNA-activated Protein kinase R (PKR) is an important and rapidly evolving Antiviral kinase. Most poxviruses contain two distinct PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), the prototypic orthopoxvirus. E3 prevents PKR homodimerization by binding double-stranded RNA, while K3 acts as a pseudosubstrate inhibitor by binding directly to activated PKR and thereby inhibiting interaction with its substrate eIF2α. In our study here, we analyzed E3 and K3 orthologs from the phylogenetically distinct capripoxviruses (CaPVs), which include lumpy skin disease virus, sheeppox virus, and goatpox virus. Whereas the sheeppox virus E3 ortholog did not substantially inhibit PKR, all three CaPV K3 orthologs showed species-specific inhibition of PKR, with strong inhibition of sheep, goat, and human PKR but only weak inhibition of cow and mouse PKR. In contrast, VACV K3 strongly inhibited cow and mouse PKR but not sheep, goat, or human PKR. Infection of cell lines from the respective species with engineered VACV strains that contained different K3 orthologs showed a good correlation of PKR inhibition with virus replication and eIF2α phosphorylation. Our results show that K3 orthologs can have dramatically different effects on PKR of different species and indicate that effective PKR inhibition by K3 orthologs is crucial for virus replication.
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Characterization of a ranavirus inhibitor of the Antiviral Protein kinase PKR.
BMC microbiology, 2011Co-Authors: Stefan Rothenburg, V. Gregory Chinchar, Thomas E. DeverAbstract:Background Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted Protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α. In analogy to distantly related Proteins found in poxviruses vIF2α might act as an inhibitor of the Antiviral Protein kinase PKR.
Guangxia Gao - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of rna recognition by zinc finger Antiviral Protein
Cell Reports, 2020Co-Authors: Xiu Luo, Xinlu Wang, Yina Gao, Jingpeng Zhu, Songqing Liu, Guangxia Gao, Pu GaoAbstract:Summary Zinc-finger Antiviral Protein (ZAP) is a host Antiviral factor that specifically restricts a wide range of viruses. ZAP selectively binds to CG-dinucleotide-enriched RNA sequences and recruits multiple RNA degradation machines to degrade target viral RNA. However, the molecular mechanism and structural basis for ZAP recognition of specific RNA are not clear. Here, we report the crystal structure of the ZAP N-terminal domain bound to a CG-rich single-stranded RNA, providing the molecular basis for its specific recognition of a CG dinucleotide and additional guanine and cytosine. The four zinc fingers of ZAP adopt a unique architecture and form extensive interactions with RNA. Mutations of both Protein and RNA at the RNA-ZAP interacting surface reduce the in vitro binding affinity and cellular Antiviral activity. This work reveals the molecular mechanism of ZAP recognition of specific target RNA and also provides insights into the mechanism by which ZAP coordinates downstream RNA degradation processes.
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trim25 is required for the Antiviral activity of zinc finger Antiviral Protein
Journal of Virology, 2017Co-Authors: Xiaojiao Zheng, Xinlu Wang, Qin Wang, Zusen Fan, Guangxia GaoAbstract:Zinc finger Antiviral Protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing Protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the Antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's Antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its Antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the Antiviral activity of ZAP is regulated.IMPORTANCE ZAP is a host Antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its Antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the Antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the Antiviral activity of ZAP is regulated.
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sindbis virus can exploit a host Antiviral Protein to evade immune surveillance
Journal of Virology, 2016Co-Authors: Xinlu Wang, Charles M. Rice, Margaret R Macdonald, Jing Zhao, Xiang Gao, Guangxia GaoAbstract:Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of Antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger Antiviral Protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs, repressing the translation of target mRNA, and promoting the degradation of target mRNA. We report here that murine embryonic fibroblast cells from ZAP knockout mice supported more efficient SVNI replication than wild-type cells. SVNI infection of 10-day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23-day-old weanling mice, whose immune system is more developed than that of the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor-neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wild-type mice. These results uncover a mechanism by which SVNI exploits a host Antiviral factor to evade innate immune surveillance. Importance Sindbis virus, a prototypic member of the Alphavirus genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host Antiviral factor that potently inhibits Sindbis virus replication in cell culture. We show here that infection of ZAP knockout suckling mice with an SVNI led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host Antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.
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structure of n terminal domain of zap indicates how a zinc finger Protein recognizes complex rna
Nature Structural & Molecular Biology, 2012Co-Authors: Shoudeng Chen, Xinlu Wang, Guangxia Gao, Kuo Zhang, Jian Sun, Yingfang LiuAbstract: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. Structural analysis now reveals a large RNA-binding surface comprising many positively charged residues and two cavities, thereby providing insight into how ZAP recognizes its target 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.
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the zinc finger Antiviral Protein recruits the rna processing exosome to degrade the target mrna
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Xuemin Guo, Jing Sun, Guangxia GaoAbstract: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.