Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.     

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.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Cap accessibility correlates with the initiation efficiency of alfalfa mosaic virus RNAs

The rate of cap removal from the alfalfa mosaic virus (A1MV) RNAs with tobacco acid pyrophosphatase (TAP) depends on the RNA species. At 37 degrees C and in the absence of divalent cation, RNA 3 reacts more slowly than the other three, which are decapped at similar rates. In the presence of magnesium, at 25 degrees C, TAP also discriminates against RNA 1. Thus the order of reactivity with TAP largely mimics the hierarchy of initiation efficiencies of the A1MV RNAs (Godefroy-Colburn et al., preceding paper in this journal). Our interpretation of these findings is that cap accessibility is what limits the rate of reaction with initiation factors as well as with TAP. In this hypothesis, translational discrimination between naturally capped messages would be related to the rate of 'breathing' of their 5' ends.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Complete nucleotide sequence of RNA 3 from alfalfa mosaic virus, strain S

We report the sequence of RNA 3 from strain S of Alfalfa mosaic virus (2,055 nucleotides). This RNA codes for a 32.4 kd protein (P3) and for the 24 kd coat protein (P4). The largest part of the sequence was established using RNA sequencing methods. The completion of the sequence in the region coding for P3 was achieved with cloned cDNA synthesized after priming at internal sites of RNA 3. Comparison of the RNA sequences coding P3 and P4 proteins in strain S with those reported in the literature for strain 425 revealed a higher amino acid substitution rate (3%) for P3 than for P4 (congruent to 1%) despite a similar average base substitution of 3-4% in these regions. In P3, two out of nine amino acid changes occur in hydrophilic regions. The amino acid changes in P4 do not modify the local hydrophilicity distribution. The intercistronic region displays a low degree of base substitution (2%) when compared with the untranslated 3'-end region (3.6%) or the 5'-end leader region (8%), the average substitution rate being 3.2%.     

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.     

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.     

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.     

Influence of a few coat protein subunits on the base-paired structure of 3'-terminal fragments of RNA 4 of alfalfa mosaic virus.

In contrast to expectation (Srinivasan, S. and Jaspars, E.M.J. (1978) Biochim. Biophys. Acta 520, 237-241) differentiated thermal melting profiles and fluorescence measurements show that the coat protein of alfalfa mosaic virus has a negligible effect on the base-paired structure of isolated 3'-terminal fragments (length about 90 nucleotides) of the coat protein messenger RNA (RNA 4) of this virus.     

Monocistronic translation of alfalfa mosaic virus RNAs.

The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems.     

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.