Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP.The PAP cDNA was placed under control of the galactoseinducible GAL] promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast.The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.Pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP) isolated from the leaves or seeds of Phytolacca americana, catalytically removes a specific adenine residue from a highly conserved stem-loop structure in the 28S rRNA of eukaryotic ribosomes (1, 2). This lesion interferes with elongation factor 2 binding and blocks protein synthesis. PAP was discovered when pokeweed extracts were found to inhibit the transmission of tobacco mosaic virus and was subsequently demonstrated to be equally effective against a number of other plant viruses (3). Studies that compare the relative antiviral properties of a number of RIPs showed that all of the RIPs tested had antiviral activity, but none was as effective as PAP (4). PAP has been shown to inhibit infection of both Vero and HeLa cells by herpes simplex virus (5) and to inhibit human immunodeficiency virus 1 (HIV-1) replication in T cells and macrophages infected in vitro at concentrations that do not inhibit cellular protein synthesis (6). A number of recent studies have shown that conjugating PAP with monoclonal antibodies dramatically increases its potency against cells infected with HIV and human cytomegalovirus (7). In addition, PAP has been used as the cytotoxic moiety of immunotoxins against acute lymphoblastic leukemia and PAPcontaining immunotoxins have shown significant antileukemic activity in clinical trials (7).Single-chain RIPs (type I RIPs), like PAP, are poorly characterized at the molecular level compared to the type II RIP, ricin, which consists of an enzymatically active...

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or Ul strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS,432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and singlestranded RNAs (up to =2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 ± 1.4 jpg/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.Tobacco mosaic virus (TMV) particles are extremely stable and retain infectivity for decades. Over 2100 copies of a 17.6-kDa coat protein (CP) fully protect the 6.4-kb singlestranded RNA (ssRNA) genome against degradation by RNases. TMV was the model system of choice for early studies on the spontaneous "self-assembly" of multimeric, biological structures in vitro (1-3). TMV assembly is initiated by a specific interaction between a prefabricated (20 S) protein aggregate (the "disk" or protohelix) and an RNA sequence centered either 0.4 kb or 0.9 kb from the 3' end of the genomic RNA (4). An origin-of-assembly sequence (OAS) was characterized (5) and proposed to exist as three stem-loop structures, before the complete 6395-nt genome of the common (or Ul) strain of TMV had been sequenced (6). The structures ofthe prefabricated, oligomeric forms ofTMV CP used for efficient assembly initiation and/or bidirectional elongation, and the precise assembly pathway (i.e., which CP form is used in which RNA direction, and whether simultaneously or sequentially) remain controversial (7)(8)(9), and the data continue to evade a consensus model (10). Two simple protocols exist to isolate CP from virions for subsequent encapsidation of ssRNA in vitro (11,12).In addition to native TMV RNA, or 3' coterminal nested genome fragments, TMV CP was shown to package chimeric ssRNAs efficiently (13, 14) in a length-and sequenceindependent manner, provided that a contiguous viral s...

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.

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