Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

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. 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. Pokeweed antiviral protein (PAP) 1The abbreviations used are: PAP, pokeweed antiviral protein; BMV, brome mosaic virus; PEG, polyethylene glycol; MES, 4-morpholineethanesulfonic acid. is a 29-kDa ribosome-inactivating protein of the pokeweed plant Phytolacca americana. Since its initial description as an antiviral agent against tobacco mosaic virus (1Duggar B.M. Armstrong J.K. Ann. Mo. Bot. Gard. 1925; 12: 359-366Crossref Google Scholar), PAP has been demonstrated to reduce the propagation of several plant and animal viruses, including potato virus X, HIV, and influenza (2Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar, 3Zarling J.M. Moran P.A. Haffar O. Sias J. Richman D.D. Spina C.A. Myers D.E. Kuebelbeck V. Ledbetter J.A. Uckun F.M. Nature. 1990; 347: 92-95Crossref PubMed Scopus (195) Google Scholar, 4Tomlinson J.A. Walker V.M. Flewtett T.H. Barclay G.R. J. Gen. Virol. 1974; 22: 225-232Crossref PubMed Scopus (88) Google Scholar). It therefore holds promise as a broad-spectrum antiviral agent. Years after its initial discovery, the enzymatic activity of PAP was characterized as an N-glycosylase (5Endo Y. Tsurugi K. Lambert J.M. Biochem. Biophys. Res. Commun. 1988; 150: 1032-1036Crossref PubMed Scopus (177) Google Scholar). Like all ribosome-inactivating proteins, PAP efficiently removes a conserved adenine from the sarcin/ricin loop within domain VI of the large ribosomal RNA (6Endo Y. Tsurugi K. J. Biol. Chem. 1987; 262: 8128-8130Abstract Full Text PDF PubMed Google Scholar, 7Stirpe F. Bailey S. Miller S.P. Bodley J.W. Nucleic Acids Res. 1988; 16: 405-412Crossref Google Scholar). This depurination slows the elongation step of protein synthesis and is considered to be the reason for cytotoxicity of the protein (reviewed in Refs. 8Wang M. Hudak K.A. Genet. Eng. (N. Y.). 2003; 25: 143-161PubMed Google Scholar and 9Tumer N.E. Hudak K. Di R. Coetzer C. Wang P. Zoubenko O. Curr. Top. Microbiol. Immunol. 1999; 240: 139-158PubMed Google Scholar). The accompanying decline in cellular protein translation may cause local cell death and limit virus propagation (10Ready M.P. Brown D.T. Robertus J.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 84: 5053-5056Crossref Scopus (118) Google Scholar). This model is supported by observations showing a positive correlation between ribosome depurination and inhibition of virus infection (11Taylor S. Massiah A. Lomonossoff G. Roberts L.M. Lord J.M. Hartley M. Plant J. 1994; 5: 827-835Crossref PubMed Scopus (106) Google Scholar). The accompanying decline in cellular protein translation, as a result of depurination, is often cited as the cause of antiviral activity. For example, reduction of poliovirus infection of HeLa cells incubated with PAP was attributed to inhibition of translation in virus-infected cells (12Ussery M.A. Irvin J.D. Hardesty B. Ann. N. Y. Acad. Sci. 1977; 284: 431-440Crossref PubMed Scopus (68) Google Scholar). In addition, inhibition of tobacco mosaic virus multiplication in tobacco protoplasts correlated well with PAP-mediated inhibition of translation (13Watanabe K. Kawasaki T. Sako N. Funatsu G. Biosci. Biotech. Biochem. 1997; 61: 994-997Crossref PubMed Scopus (19) Google Scholar). More recent results have revealed that many ribosome-inactivating proteins are capable of depurinating RNA substrates apart from the rRNA (14Bolognesi A. Polito L. Lubelli C. Barbieri L. Parente A. Stirpe F. J. Biol. Chem. 2002; 277: 13709-13716Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Wang P. Tumer N.E. Nucleic Acids Res. 1999; 27: 1900-1905Crossref PubMed Scopus (59) Google Scholar). Rajamohan et al. (17Rajamohan F. Venkatachalam T.K. Irvin J.D. Uckun F.M. Biochem. Biophys. Res. Commun. 1999; 260: 453-458Crossref PubMed Scopus (84) Google Scholar) showed that PAP removes both adenines and guanines from HIV-1 when incubated in vitro with the genomic viral RNA. In addition, Hudak et al. (18Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar) have shown that PAP and nontoxic PAP mutants depurinate brome mosaic virus (BMV) RNAs in vitro and that this depurination inhibits their translation in a cell-free system. Therefore, the direct depurination of viral RNAs by PAP may contribute to its antiviral activity. BMV is a model positive-strand RNA virus with a genome composed of three positive sense RNAs designated RNA1, RNA2, and RNA3. Each RNA is 5′-capped and contains a conserved 200-nucleotide tRNA-like structure at the 3′-end (reviewed in Refs. 19Ahlquist P. Curr. Opin. Genet. Dev. 1992; 2: 71-76Crossref PubMed Scopus (139) Google Scholar and 20Kao C.C. Sivakumaran K. Mol. Plant Pathol. 2000; 1: 91-97Crossref PubMed Scopus (83) Google Scholar). RNA1 is monocistronic and encodes a 1a protein containing an N-terminal domain with similarity to m7G methyltransferases involved in viral RNA capping and a C-terminal domain with similarity to RNA helicases (21Kong F. Sivakumaran K. Kao C.C. Virology. 1999; 259: 200-210Crossref PubMed Scopus (64) Google Scholar, 22Ahola T. den Boon J.A. Ahlquist P. J. Virol. 2000; 74: 8803-8811Crossref PubMed Scopus Google Scholar). is monocistronic and encodes a protein that has all of the of RNA P. Nucleic Acids Res. 1988; 16: PubMed Scopus Google Scholar). RNA3 is and encodes a protein and a protein that is from a subgenomic RNA4 Nature. PubMed Scopus Google Scholar, R. C. Ahlquist P. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar, Virology. PubMed Scopus Google Scholar). of the RNAs therefore replication of and positive RNAs and of subgenomic RNA. In this we expand on initial in vitro studies to show that PAP and nontoxic PAP mutants the replication and of BMV RNAs in barley The inhibition by PAP is to ribosome depurination decline of cellular translation. PAP and nontoxic mutants reduced the accumulation of BMV RNAs in by both viral RNA replication and transcription. depurinated RNAs shown to RNA synthesis by the BMV replicase in and of in E. of PAP was from a the of The was to at and the was to at the of the protein in The mutants of PAP, namely and with the and all into the at and by and into The and of PAP by on a containing PAP and by with a proteins by and with with of PAP and PAP mutants from E. an was from et al. K. L. Kao C.C. J. Virol. PubMed Scopus Google Scholar). RNA template of was with and of RNA was incubated with of PAP PAP mutants in and at for RNA incubated protein was used as a and the of the was used as a positive K. L. Kao C.C. J. Virol. PubMed Scopus Google Scholar). The positive was incubated in the as the that was with by a The was in and of was using a of and of isolated from barley to the by Tumer et al. (2Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). PAP and PAP mutants from E. coli incubated with barley in to a of for at rRNA was and depurination was by as previously (18Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar) using of barley to the of the was in for and as an for RNA Coetzer C. Tumer N.E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of BMV RNAs with PAP and PAP RNAs isolated from viral incubated with PAP in to a of for at PAP was from the RNAs by and the RNAs in The RNAs in and used to isolated protoplasts of BMV RNAs with PAP mutants as for with in vitro of BMV RNA3 was incubated with PAP as for BMV of and with BMV by of of and of protoplasts with BMV RNAs was using to the of et al. P. P. Ahlquist P. J. Virol. PubMed Google Scholar). BMV RNA as to protoplasts in and incubated for at and in and in of incubated for at and a of BMV RNAs the of PEG, and the positive was of BMV RNAs in the of prior with of with PAP and PAP of protoplasts with of BMV RNAs and incubated as in of for prior to the of PAP incubated for an at and the effect of PAP to protoplasts on the synthesis of positive BMV RNAs, of protoplasts with of BMV RNAs and as for prior to the of of PAP and for a of PAP a decline in the of BMV RNAs in a of protoplasts was with of in vitro of BMV RNA1 and incubated in of for prior to the of of at the and for the of BMV RNA1 by protoplasts the of PAP used as a of RNA and of RNA was isolated from these at for and in MES, and and at for The was with and RNA was by the of of and of The RNA was by and with with and in of RNA in a to and with of BMV The for positive-strand BMV RNA was from containing an of the tRNA-like structure from the 3′-end of BMV RNA3 that is conserved in in all BMV positive The for BMV RNA was from the in the The rRNA was from containing an of the conserved sarcin/ricin loop of the of to RNA was by of the to In synthesis in protoplasts was by the of into with BMV RNAs for in and incubated in of containing of PAP PAP mutants for at with of and at the by at and of was to The was incubated at for by on was with and and was by BMV used in replicase from BMV replicase was isolated from barley as by et al. J. S. G. Kao C.C. Virology. PubMed Scopus Google Scholar). to et al. S. S. G. R. Kao C.C. RNA. 1998; Google Scholar). template RNA and of replicase in in a at for the with and in of of and a of in and on The of into RNA was with a and using the of RNA synthesis the replicase was as and of at the and as The of synthesis for to the template in the of of PAP and PAP in E. PAP is as a that is to the of the has and from the and of the in vitro activity of PAP has been by E. of the protein F. Uckun F.M. 1999; 16: PubMed Scopus Google Scholar, E. K. Biosci. Biochem. 1999; PubMed Scopus Google Scholar). of PAP from E. coli have been of PAP and its mutants in E. coli the synthesis of their their synthesis in The and mutants with to at and with to proteins of these proteins an N-terminal of PAP in to is an with a that the activity of this protein Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (56) Google Scholar). and a and a in of and are nontoxic to Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (56) Google Scholar). The proteins to their with to the of C-terminal the proteins and, at the of PAP used in protoplast assays, activities showed the of of the accumulation of BMV RNAs in protoplasts and was in this was to a of all proteins isolated in the of BMV RNA accumulation in barley protoplasts by prior with PAP and PAP BMV RNAs incubated with PAP and PAP was by The BMV RNAs into protoplasts and to for protoplast RNA was by and for positive BMV protoplast and a BMV RNAs used as for BMV RNAs incubated in prior to into protoplasts used as a positive for of BMV RNAs directly the the for rRNA as a for RNA. BMV RNAs incubated with PAP, and by The BMV RNAs into protoplasts and to for protoplast RNA was by and for positive BMV BMV RNAs incubated in prior to into protoplasts used as a positive for of BMV RNAs directly the the for rRNA as a for of of PAP and PAP mutants with barley protoplasts with BMV protoplasts with of BMV RNAs with and incubated in of containing of PAP PAP mutants for protoplast RNA was by and for positive BMV incubated PAP used as a positive for BMV of BMV RNAs directly the the for rRNA as a for RNA. protoplasts with of BMV RNAs with and incubated in of for prior to the of incubated for a of protoplast RNA was by and for positive and BMV incubated PAP used as a positive for BMV the for rRNA as a for of by E. PAP and PAP mutants and depurinate when in tobacco O. Hudak K.A. Tumer N.E. Plant Mol. Biol. 2000; PubMed Scopus (56) Google Scholar, K.A. J. Tumer N.E. J.D. Virology. PubMed Scopus Google Scholar). PAP and PAP mutants in E. coli able to depurinate the proteins incubated with isolated from barley and was on the rRNA to the missing is a for BMV, the of this shown in only of depurination of the sarcin/ricin loop in barley with This depurination may be to the ribosome-inactivating protein in barley B. V. S. K. J. Plant J. 1994; 6: PubMed Scopus Google Scholar). of with PAP from pokeweed rRNA depurination levels. of depurination for incubated with PAP isolated from E. In the nontoxic mutants and depurinate barley levels. These that the of PAP in E. coli depurination as the proteins in tobacco and of BMV RNA in by Pretreatment with have demonstrated previously that PAP BMV RNAs in vitro and inhibits the translation of these RNAs in a cell-free (18Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). PAP BMV RNAs in the RNAs incubated with PAP, by and to RNAs into barley and the of replication that was by BMV RNAs of replication are by this a decline in the of BMV RNAs that correlated with of of These results that prior of BMV RNAs with PAP inhibits the accumulation of these RNAs in barley This was with of nontoxic mutants of PAP, an that severe inhibition of accumulation with PAP that both and able to efficiently accumulation of BMV, for rRNA The BMV RNA the of viral RNA was from PAP These results that rRNA depurination and inhibition of BMV RNA accumulation both PAP with a has as revealed by mutants and for rRNA as an of RNA and of PAP in PAP BMV RNA accumulation in prior of the viral RNAs, PAP PAP mutants to the protoplast after RNA PAP is to be able to protoplast of PAP to the was (13Watanabe K. Kawasaki T. Sako N. Funatsu G. Biosci. Biotech. Biochem. 1997; 61: 994-997Crossref PubMed Scopus (19) Google Scholar). of protoplast RNA after an a decline in the of BMV RNAs for with PAP, The of BMV RNA accumulation was to that when the RNAs incubated with PAP PAP mutants prior to the transfection of BMV in vitro and in with PAP have on BMV accumulation in barley for rRNA as a for RNA we PAP the replication of positive BMV RNAs after the of RNA after BMV translation and RNA replication are to be well positive RNA synthesis is M. Kao C.C. J. Virol. PubMed Scopus Google Scholar). that PAP at transfection the accumulation of positive RNAs to the to the accumulation in the of PAP the accumulation of RNA. Therefore, synthesis of positive RNA is more to the of PAP RNA. RNA was shown by these for rRNA PAP the of BMV RNAs in in vitro of BMV RNA1 was into protoplasts and incubated in to PAP was of protoplasts a and for the of BMV This in protoplasts for and was by at after transfection This was by the of PAP, that PAP the of this in protoplasts for rRNA as a for RNA and the decline in BMV RNA accumulation PAP was to an inhibition of protein barley protoplasts incubated with PAP PAP and the of a of was a the of to the protoplasts incubated with PAP a of with cells incubated in This result that PAP in the is able to the of protoplasts to translation. the cause a reduction in protein the translation of protoplasts incubated with was with the of in vitro depurination of barley Therefore, the reduction in accumulation of BMV RNAs in protoplasts incubated with was to inhibition of protein of PAP on RNA are several at BMV RNA accumulation be as in of BMV RNA results show that and cause a decline in translation of barley BMV RNA accumulation be at the template that we have previously shown that PAP can the translation of BMV RNAs by direct depurination of the viral RNAs (18Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). We have inhibition of positive BMV RNA accumulation to we that PAP activity on BMV RNAs viral replication transcription. BMV RNA synthesis can be into the of RNA the synthesis of genome and positive RNAs, and the synthesis of subgenomic PAP replication in vitro of of the three BMV RNAs and only BMV RNA3 was with PAP prior to of all RNAs into We that BMV RNA1 and can in the of RNA3 for replication M. Kao C.C. J. Virol. PubMed Scopus Google Scholar). a in the of BMV RNA3 an of protoplasts in RNA1 and to the protein from RNA3 the accumulation of RNA1 and C.C. Virology. PubMed Scopus Google Scholar). In protoplasts with RNAs with PAP, subgenomic from a was efficiently RNA4 was in protoplasts with BMV RNA3 that of BMV RNA3 with PAP subgenomic to genomic RNA for rRNA as an of RNA of RNA on the BMV results from of RNA3 that PAP a at the of RNA replication transcription. demonstrate the of this mechanism of we the effect of template depurination on RNA synthesis in The containing depurinated at and used for RNA synthesis by the BMV The is from a well characterized RNA and can direct from the in the Kao C. Biol. 2000; PubMed Scopus (64) Google Scholar) Since nucleotide only in the at the of containing the at at both and and their to template RNA synthesis was These RNA at with the RNA with and a at the was a in we at and in and three to the within Therefore, within RNA the BMV replicase to RNA synthesis at these In addition, the synthesis of RNA in to the the For example, the of was of the RNA for with was of the RNA. These results that the of the BMV replicase to RNA synthesis at on the of the et al. J. S. G. Kao C.C. Virology. PubMed Scopus Google Scholar) and and Kao Kao C. Virology. 1997; PubMed Scopus Google Scholar) previously that the BMV replicase to elongation after the replicase has between and RNA may cause the replicase to stall on the RNA this a of was to the of from with in RNA synthesis from to that with template with Therefore, depurination of RNA within of the the BMV replicase to depurination causes the replicase to stall on the RNA. These results demonstrate that depurination of BMV RNA by PAP inhibits the synthesis of and of the viral replicase is on the of the within the template RNA depurination by PAP at a after is that the replicase be from In this we have and several of PAP from E. coli and shown that the as their the mutants and to depurinate barley rRNA in vitro and of cellular protein synthesis in vivo. and the to virus accumulation in protoplasts an effect on cellular translation. of these proteins and their activities novel of PAP on virus namely inhibition of distinct of viral RNA with these with RNA synthesis by the BMV replicase in of BMV RNAs with PAP mutants the accumulation of these viral RNAs in barley of that this inhibition is to PAP activity. was a correlation between PAP and the of BMV the effect on BMV The that from E. coli the inhibition is the was as the of These that the PAP was for the inhibition and the results protoplasts as an for PAP-mediated in cells and in using tobacco and potato virus X, demonstrated the of PAP on viral infection (2Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). The show that PAP and its are in a and are against a RNA this the of the of PAP against a of that of the for PAP activity against has been on (12Ussery M.A. Irvin J.D. Hardesty B. Ann. N. Y. Acad. Sci. 1977; 284: 431-440Crossref PubMed Scopus (68) Google Scholar, K. Kawasaki T. Sako N. Funatsu G. Biosci. Biotech. Biochem. 1997; 61: 994-997Crossref PubMed Scopus (19) Google Scholar). of depurinate was to virus in (2Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). We show that PAP mutants cause reduction in cellular translation that an mechanism is involved in antiviral activity. studies have shown that of BMV RNAs in vitro with PAP to depurination of the and inhibition of their translation in a cell-free (18Hudak K.A. Wang P. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). Therefore, the inhibition of BMV RNA accumulation in protoplasts with PAP in be to inhibition of BMV RNA translation. the demonstrated of PAP to depurinate BMV RNAs at in the BMV For example, the inhibition of positive RNA accumulation a for PAP that PAP inhibits the replication of virus the cellular the BMV genome to proteins 1a and involved in viral proteins for replication of viral RNA to The that PAP the accumulation of positive RNAs that translation inhibition is the reason for of RNA that this both and that of BMV RNA3 inhibits accumulation of BMV RNAs in protoplasts is with inhibition at the of RNA of protein is for BMV RNA and studies have shown that the of these proteins BMV RNA accumulation in protoplast R. Ahlquist P. J. Virol. 1987; 61: PubMed Google Scholar). Therefore, inhibition of BMV RNA accumulation is with a PAP-induced in viral RNA replication of inhibition of protein The reduction of all three genomic BMV RNAs can be by the activity that RNA3 has on RNA1 and C.C. Virology. PubMed Scopus Google Scholar). Since PAP is to depurinate BMV RNAs in vitro, the for PAP inhibition of viral RNA replication is the of more in the RNA are to synthesis by Genet. 1986; PubMed Scopus Google Scholar, M. S. J. PubMed Scopus Google Scholar), and in vitro results with the BMV replicase show the to be for this RNA The inhibition of RNA accumulation in protoplasts PAP of BMV RNA3 be to an effect on the of a in the of RNA by the PAP with protoplasts with BMV RNA1 the of the to PAP Therefore, we have that PAP the of BMV in vivo. the mechanism is supported by that an to the nucleotide in of the to an within the template et al. J. S. G. Kao C.C. Virology. PubMed Scopus Google Scholar) and and Kao Kao C. Virology. 1997; PubMed Scopus Google Scholar, Kao C. Virology. 1997; PubMed Scopus Google Scholar) previously demonstrated that the BMV replicase synthesis and to of the after the replicase has more of the is more with the template RNA Kao C. Virology. 1997; PubMed Scopus Google Scholar, Kao C. Virology. 1997; PubMed Scopus Google Scholar). It is viral RNA can from a as been demonstrated for RNA F. C. J. A. M. J. 2000; PubMed Scopus Google Scholar). These observations be to the of RNA of this was that of the positive RNA3 with PAP in inhibition of subgenomic RNA4 synthesis in with RNA3. RNA4 by at a in RNA3 R. M. Ahlquist P. 1986; PubMed Scopus Google Scholar, E. N. Ahlquist P. Virology. 1994; PubMed Scopus Google Scholar, K. M. Kao C. J. Virol. PubMed Scopus Google Scholar). The that RNA3 was able to in these that RNA3 synthesis may have from subgenomic RNA4 transcription. the in PAP in vitro of all three BMV RNAs cause inhibition of subgenomic RNA4 transcription. We the for the inhibition of RNA4 synthesis when only RNA3 was with PAP, the effect was in three of the in vitro and in activities of PAP, we have for novel by PAP can viral distinct in the virus by PAP RNA replication and subgenomic RNA transcription. These only the antiviral activities of PAP, provide viral targets for viral studies on a of the involved in these novel and the to contribute to viral inhibition in vivo. We K. A. for of the

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