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.

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.

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.

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.

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.

Protein binding sites in nucleation complexes of alfalfa mosaic virus RNA 4.

The subgenomic coat protein messenger RNA 4 of alfalfa mosaic virus forms complexes with one and three coat protein dimers, which are designated complexes I and III, respectively. These complexes were separated, subjected to digestion with ribonuclease T1, and filtered onto Millipore filters. Phenol extracts of the filters contained specific fragments of RNA 4, which were sequenced after electrophoretic separation on nondenaturing and denaturing polyacrylamide gels. Complex I yielded only a 68-nucleotide fragment including the 3' terminus [fragment 814-881 according to the numbering of Brederode, F. Th., Koper-Zwarthoff, E. C., & Bol, J. F. (1980) Nucleic Acids Res. 8, 2213-2223]. Complex III yielded in addition to the former fragment also other, mostly extracistronic, fragments from the 3'-terminal region, as well as fragments from an intracistronic region, comprising positions 425-474, in the middle of RNA 4. The 3'-terminal region was subdivided by small gaps into three coat protein binding sites: 799-881, 759-787, and 667-753, designated sites 1, 2, and 3, respectively, and possibly representing the sites occupied by the three coat protein dimers. A similarity may exist between the secondary structure of sites 1 and 3, which both may have three hairpins, two of which flanked at their 3' side by an AUGC sequence. Furthermore, a complementarity was noted between the loop of a large hairpin which can be drawn in the intracistronic site and the upper part of one of the three hairpins in the 3'-terminal site 1. These binding features have been combined in a model structure for the complex of RNA 4 with three coat protein dimers.

Preferential binding of 3'-terminal fragments of alfalfa mosaic virus RNA 4 to virions.

3'-Terminal fragments present in a partial ribonuclease T1 digest of RNA 4 of alfalfa mosaic virus were selectively bound to virions of the same virus by incubation in 10 mM sodium phosphate and 1 mM EDTA, pH 7.0. The virions with the associated fragments were separated from the remainder of the digest by velocity gradient centrifugation. When the incubation was performed in 27.7 mM Na2HPO4 and 6.2 mM citric acid, pH 6.8, plus 10% glycerol, much more, but less specific, binding was obtained. However, in this case the terminal fragments present in unbound material of the digest were almost exclusively 5' termini. The four RNA species species of alfalfa mosaic virus have an extensive 3'-terminal homology. Therefore, it is expected that specific virion binding can be used to obtain 3'-terminal fragments of different lengths of the three genome RNA species (RNAs 1, 2, and 3) of alfalfa mosaic virus and, possibly, of related viruses. Such fragments are of great importance for the study of the specific 3'-terminal interaction with coat protein subunits which is thought to be responsible for the activation of the viral genome.

Complexes of alfalfa mosaic virus RNA 4 with one and three coat protein dimers.

RNA 4, the subgenomic coat protein messenger of alfalfa mosaic virus, was loaded with small amounts of coat protein in a reaction in which complete virions were the protein donor. In such a reaction the protein subunits attach to the high-affinity binding sites near the 3' end of RNA 4 [Houwing, C. J., & Jaspars, E. M. J. (1978) Biochemistry 17, 2927-2933]. At a ratio of up to 13 coat protein subunits to 1 mol of RNA 4, complexes with one and three protein dimers, designated complex I and complex III, respectively, were formed. These complexes were isolated by preparative electrophoresis in 4% polyacrylamide gel. At a large excess of the protein donor (280 protein subunit/mol of complex), both complexes I and III were converted into uniform complexes with 10 protein dimers. There were no indications for stable intermediate complexes. A model is suggested for the structure of the complexes which is based on the model proposed for the protein coat of alfalfa mosaic virus [Mellema, J. E., & Van Den Berg, H. J. N. (1974) J. Supramol. Struct. 2, 17-31]. The complexes possibly serve as successive stages in virion assembly. More intriguingly, the complexes could be of regulatory significance. Since the four RNA species of alfalfa mosaic virus have an extensive 3'-terminal homology, and since 3'-terminal interaction with coat protein subunits is thought to be a process leading to recognition of the viral genome by the viral replicase and thus to infectivity, complexes analogous to complexes I and III could represent the infectious forms of the genome RNAs.

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.