A genome-activating N-terminal coat protein peptide of alfalfa mosaic virus is able to activate infection by RNAs 1, 2 and 3 but not by RNAs 1 and 2. Further support for the messenger release hypothesis.

An N-terminal genome-activating peptide of 25 amino acid residues of alfalfa mosaic virus coat protein was unable to activate the incomplete viral genome consisting of RNAs 1 and 2. The messenger release hypothesis predicts that RNA 3 must complement such an inoculum in order to produce RNA 4 that will trigger the process. This is shown indeed to be the case.

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis.

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

Genome activation in alfamo- and ilarviruses.

Alfamo- and ilarviruses are characterized by the deficiency of their genomes (three messenger-sense RNAs) to start an infection cycle. The RNAs are in capsids built from a single species of protein of about 24 kD. A few dimers of this coat protein per RNA molecule are sufficient to activate the genome. Since the first description of genome activation [Bol JF, van Vloten-Doting L, Jaspars EMJ (1971) Virology 46: 73-85] three models have been proposed concerning its mechanism: the protection, the replicase and the messenger release hypotheses. The first two models make use of the fact that in these genera of RNA viruses the 3' termini of the RNAs bind the coat protein very strongly. The resulting structure would provide protection against 3'- to 5' exoribonucleases, or would permit correct initiation of minus-strand synthesis, respectively. However, naked inoculated RNAs of alfalfa mosaic virus appear to be quite stable in the cell, and in vitro the coat protein is inhibiting rather than stimulating initiation of minus-strand synthesis. The messenger release hypothesis states that the coat protein is needed for the release of viral messenger RNAs from membranous replication complexes throughout the whole viral replication cycle. This is supported by in vivo and in vitro observations, but as yet a detailed molecular mechanism is difficult to give.

Regulation of single-strand RNA synthesis of alfalfa mosaic virus in non-transgenic cowpea protoplasts by the viral coat protein.

We have compared the RNA synthesis of alfalfa mosaic virus in complete (by RNAs 1, 2 and 3) and incomplete infections (by RNAs 1 and 2) of cowpea protoplasts. Both viral RNA polymerase activity and accumulation of viral RNA were measured. By annealing RNA in solution with 32P-labelled probes of plus and minus polarity followed by treatment with ribonucleases, we determined viral RNAs quantitatively in both single- and double-stranded RNA fractions. The accumulation of single-stranded RNA of positive polarity differed considerably between the two types of infection (250 ng vs. less than 1 ng per 10(5) protoplasts), although viral RNA polymerase activities as measured in vitro and the concentrations of minus RNA were similar. Since the method also measured fragmented RNA, this difference is probably not due to lack of protection of viral RNA by coat protein during incomplete infection. Synthesis of single-stranded plus RNA requires either RNA 3 itself or one of its gene products. We postulate that coat protein is the stringent regulator of alfalfa mosaic virus genomic expression.

A core promoter hairpin is essential for subgenomic RNA synthesis in alfalfa mosaic alfamovirus and is conserved in other Bromoviridae.

The nucleotide sequence immediately in front of the initiation site for subgenomic RNA 4 synthesis on RNA 3 minus strand, which has been proved to function as a core promoter, was inspected for secondary structure in 26 species of the plant virus family Bromoviridae. In 23 cases a stable hairpin could be predicted at a distance of 3 to 8 nucleotides from the initiation site of RNA 4. This hairpin contained several conserved nucleotides that are essential for core promoter activity in brome mosaic virus (R.W. Siegel, S. Adkins and C.C. Kao, Proc. Natl. Acad. Sci. USA 94, 11238-11243, 1997). Phylogenetic evidence and evidence from the effect of artificial mutations reported in the literature (E.A.G. van der Vossen, T. Notenboom and J.F. Bol, Virology 212, 663-672, 1995) indicate that the stem-loop structure is essential for promoter activity in alfalfa mosaic virus and probably in other Bromoviridae. Stability of the hairpin is most pronounced in the genera Alfamovirus and Ilarvirus which display genome activation by coat protein. The hypothesis is put forward that with these viruses the coat protein is needed for the viral RNA polymerase to interact with the core promoter hairpin leading to access for the enzyme to the initiation site of RNA 4.

Efficient transcription of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase obtained by a new procedure [corrected].

The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion with ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66.

Interaction between RNA-dependent RNA polymerase of alfalfa mosaic virus and its template: oxidation of vicinal hydroxyl groups blocks in vitro RNA synthesis.

In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependent in vitro RNA synthesizing system of alfalfa mosaic virus (AIMV) we were able to study the effect of small chemical modifications of the 3' end of the template RNAs on product formation. After oxidation of the 3'-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuyl et al., Virology 176, 346-354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3' end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AIMV displays a high specificity for its cognate template, either before or after the oxidation of the 3'-terminal nucleoside. From this it was concluded that periodate modification of the 3'-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadt et al., Virology 182, 309-315, 1991), was not the main target involved in the inhibition of RNA synthesis.

RNA duplex unwinding activity of alfalfa mosaic virus RNA-dependent RNA polymerase.

An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic virus-infected tobacco is capable of synthesizing in vitro full-size RNAs of minus and plus polarities. However, the enzyme is not able to perform a complete replication cycle in vitro. The products were found to be completely base-paired to their templates. The enzyme was able to use double-stranded RNA as a template for RNA synthesis if it could initiate from a single-stranded promoter. The inability (of most) of our enzyme preparations to create a single-stranded initiation site could explain why they could not perform a complete replication cycle in vitro. This is the first report on duplex RNA unwinding activities by a plant viral RdRp.

In vitro evidence that the coat protein of alfalfa mosaic virus plays a direct role in the regulation of plus and minus RNA synthesis: implications for the life cycle of alfalfa mosaic virus.

The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of cost protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181, 687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minus-strand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.

Localization and biochemical characterization of alfalfa mosaic virus replication complexes.

Replication complexes were isolated from alfalfa mosaic virus-infected tobacco protoplasts. Most of the RNA-synthesizing activity appears to colocalize with the intact chloroplasts upon sucrose-gradient centrifugation of cellular homogenates. Further analysis of these replication complexes showed that the enzyme is strongly associated with the outside of the chloroplasts, the endogenous template being well protected against ribonuclease action. RNA polymerase activity is sensitive to protease treatment of intact chloroplast fraction showing that an essential part of the enzyme complex is facing the in vitro medium, and probably the cytosol in vivo.

Coat protein stimulates replication complexes of alfalfa mosaic virus to produce virion RNAs in vitro.

Viral replication complexes (RCs) were gradient-purified from cowpea mesophyll protoplasts 21 h after inoculation with alfalfa mosaic virus. These membranous structures incorporate [32P]UMP into double- and single-stranded RNAs in the absence of added template. When coat protein is added prior to the reaction the incorporation in both RNA fractions is stimulated several times. Part of the single-stranded product RNAs are released from the RCs. The stimulation of incorporation in high molecular mass RNAs by coat protein can be mimicked only to a certain extent by addition of a ribonuclease inhibitor or of an excess of viral RNA prior to the reaction. This shows that the coat protein is not only protecting the product of the RCs against degradation by ribonuclease, but that it is stimulating the synthesis and release of viral RNAs from RCs as well. This leads to the hypothesis that with alfalfa mosaic virus some coat protein is necessary for the release of messenger RNA from the RC. The hypothesis explains why the viral genome RNAs, although they are of messenger polarity, cannot start a replication cycle in the absence of coat protein: RCs containing the parental RNAs could be formed but no amplification of them could take place since no messenger RNAs needed for the production of viral polymerase proteins would be released into the cytoplasm.

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.

Analysis of the protein composition of alfalfa mosaic virus RNA-dependent RNA polymerase.

RNA-dependent RNA polymerase (RdRp) was solubilized and purified from cellular membranes isolated from alfalfa mosaic virus (AIMV)-infected tobacco by employing a procedure recently described for brome mosaic virus RdRp [R. Quadt and E.M.J. Jaspars, 1990, Virology 178, 189-194]. The purified AIMV RdRp is completely dependent on added template RNAs and exhibits a high degree of template specificity. Analysis of the protein composition of AIMV RdRp showed that AIMV-encoded proteins P1 and P2 and the coat protein (CP) are present in the active enzyme complex. Minus-strand synthesis by the AIMV RdRp is inhibited by AIMV CP. Native double-stranded AIMV RNAs are utilized as template for viral RNA synthesis by AIMV RdRp indicating that a helicase activity is present in the purified AIMV RdRp preparation.

Characterization of cucumber mosaic virus RNA-dependent RNA polymerase.

Cucumber mosaic virus (CMV) RNA-dependent RNA polymerase (RdRp) was purified form CMV-infected tobacco. The purified enzyme is completely dependent on exogenous template. The enzyme utilizes a variety of viral RNAs and CMV satellite RNA as template for minus-strand synthesis. Cellular RNAs are not used as templates. Ribosomal RNA inhibits the viral RNA synthesis by the CMV RdRp.

Effect of removal of zinc on alfalfa mosaic virus RNA-dependent RNA polymerase.

The necessity of coat protein for infection of plants by alfalfa mosaic virus (AIMV) and other ilarviruses distinguishes this virus group from other plant virus groups. Recently, the presence of both a zinc-finger type motif and zinc in AIMV coat protein was described [(1989) Virology 168, 48-56]. We studied the effect of a zinc chelator on viral RNA synthesis. Strong inhibition of AIMV RNA-dependent RNA polymerase (RdRp) by ortho-phenanthroline (OP) was observed.

Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase.

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.

cis-acting elements involved in replication of alfalfa mosaic virus RNAs in vitro.

A DNA copy of alfalfa mosaic virus (AIMV) RNA3 was transcribed in vitro in two different orientations with T7 RNA polymerase and the transcripts were used as templates for a virus-specific RNA-dependent RNA polymerase (RdRp) purified from AIMV-infected bean plants. Minus-stranded templates were transcribed by the RdRp into subgenomic plus-stranded RNA4. A deletion analysis showed that a sequence in minus-strand RNA3, located between nucleotides -8 and -55 upstream of the initiation site for RNA4 synthesis, was sufficient for subgenomic promoter activity in vitro. Plus-stranded templates were transcribed by the RdRp into full-length minus-stranded copies. A deletion analysis indicated that a sequence located between nucleotides 133 and 163 from the 3'-end of AIMV RNA3 was sufficient to direct the synthesis of minus-stranded products by the RdRp. Thus, the 3'-terminal region of the AIMV RNAs, which contains the binding sites with a high affinity for coat protein, appears not to be involved in recognition of the RNAs by the RdRp in vitro.

Involvement of a nonstructural protein in the RNA synthesis of brome mosaic virus.

RNA-dependent RNA polymerase (RdRp) was prepared from brome mosaic virus (BMV)-infected barley by a procedure including Nonidet-P40 treatment. The enzyme proved to be highly active, specific, and almost completely template dependent without the need for nuclease treatment [W. A. Miller, and T. C. Hall (1983) Virology 125, 236-241] or DEAE ion exchange chromatography [K. Maekawa and I. Furusawa (1984) Ann. Phytopathol. Soc. Japan 50, 491-499]. Two C-terminal peptides P1C and P2C derived from the nonstructural BMV proteins P1 and P2, respectively, were synthesized. Antibodies raised against these peptides were able to recognize the corresponding native proteins present in RdRp preparations. Antibodies directed against P1C were capable of completely blocking the transcription of BMV RNA in vitro. This is the first experimental evidence that a nonstructural viral protein is present in an enzyme complex involved in tricornaviral RNA synthesis.

Viral RNA synthesis by a particulate fraction from cucumber seedlings infected with cucumber mosaic virus.

Particulate preparations from cucumber mosaic virus (CMV)-infected cucumber cotyledons incorporated [alpha-32P]GTP into products which, after phenol extraction, appeared to be double-stranded forms of the four CMV RNAs. In preparations isolated 3 to 5 days after inoculation, the label was incorporated mostly into plus RNA as shown by dot-blot hybridization using single-strand recombinant DNA clones of CMV RNA. After a short pulse the labeled material consisted mostly of small heterogeneous products, but part of the label could be chased into full-length RNAs, demonstrating that the synthetic process was continuous strand elongation rather than terminal addition. CMV RNAs added to the particulate fraction did not give rise to high molecular weight transcripts. In addition to the high molecular weight defined products which remained in the particulate fraction, small heterogeneous products, complementary to plant RNA and to viral RNA of both plus and minus polarity, were released into the incubation medium. They appeared to be products of the virus-induced Mr 100,000 (100K) protein which is solubilized from the particulate fraction during incubation. However, when the particulate fraction was first subjected to an extensive solubilization and washing treatment in order to remove the M(r) 100K protein [D. S. Gill, R. Kumarasamy, and R. H. Symons (1981) Virology 113, 1-8], and then used for product synthesis, a large amount of the product was still of small size, suggesting that the synthesis of high and low molecular weight RNA was intrinsically connected, and that the M(r) 100K protein was not merely contaminating the particulate fraction.