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.     

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.     

Structural elements of the 3'-terminal coat protein binding site in alfalfa mosaic virus RNAs.

The 3'-terminal of the three genomic RNAs of alfalfa mosaic virus (AIMV) and ilarviruses contain a number of AUGC-motifs separated by hairpin structures. Binding of coat protein (CP) to such elements in the RNAs is required to initiate infection of these viruses. Determinants for CP binding in the 3'-terminal 39 nucleotides (nt) of AIMV RNA 3 were analyzed by band-shift assays. From the 5'- to 3'-end this 39 nt sequence contains AUGC-motif 3, stem-loop structure 2 (STLP2), AUGC-motif 2, stem-loop structure 1 (STLP1) and AUGC-motif 1. A mutational analysis showed that all three AUGC-motifs were involved in CP binding. Mutation of the A- and U-residues of motifs 1 or 3 had no effect on CP binding but similar mutations in motif 2 abolished CP binding. A mutational analysis of the stem of STLP1 and STLP2 confirmed the importance of these hairpins for CP binding. Randomization of the sequence of the stems and loops of STLP1 and STLP2 had no effect on CP binding as long as the secondary structure was maintained. This indicates that the two hairpins are not involved in sequence-specific interactions with CP. They may function in a secondary structure-specific interaction with CP and/or in the assembly of the AUGC-motifs in a configuration required for CP binding.     

Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (A1MV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of A1MV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of A1MV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for A1MV RNAs 1 and 2 but not for RNA 3.     

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.     

Structure of the 5'-terminal untranslated region of the genomic RNAs from two strains of alfalfa mosaic virus.

We report the sequences of the 5'-terminal regions of the 3 Alfalfa Mosaic Virus genomic RNAs for the Strasbourg strain (AlMV-S) and for a new isolate, AlMV-B; they are compared to similar data obtained by Koper-Zwarthoff et al. (Nucleic Acids Res. 1980, 8, 5635-5647) for strain 425. The structure of these leaders is highly conserved in RNAs 1 and 2. The length of the leader is 102, 100 and 101 nucleotides in RNA1 for strains S, B and 425 respectively; 55 and 56 in RNA2 for strains S and B respectively. In RNA3 however, there are important differences near the 5'-terminus between strain S and the other two: The total leader length is 258 nucleotides for strain S and 242 for strain B. The secondary structure models show a conserved hairpin near the 5'-end of each genomic RNA of AlMV-S. This hairpin is inexistent in RNA3 of the B and 425 strains. The degree of base-pairing increases with leader length. The initiator codon is located in a single stranded region in RNA2 whereas it is found in a hairpin stem in RNA 3.     

Model for the capsid structure of alfalfa mosaic virus.

The coat protein of alfalfa mosaic virus is clustered in such a way as to avoid the 3 and 6-fold lattice positions of the hexagonal surface lattice. This results in a relatively open structure for the capsid, which is mainly caused by holes situated at the 6-fold positions and, to a minor extent, those present at the 3-fold lattice points. The evidence for this has been obtained by analysing electron micrographs of negatively stained virus particles by optical diffraction and digital image processing.     

A study of the states of aggregation of alfalfa mosaic virus protein.

The states of aggregation of alfalfa mosaic virus (AMV) protein have been characterized by sedimentation velocity experiments and electron microscopy. The main association product is a spherical particle with an s value of about 30S. It is highly likely that the assembly of this particle starts with dimers of the 25000 molecular mass unit resulting in an icosahedral particle made of 30 dimers. No intermediate aggregation products have been detected. The clustering pattern of the protein in the cylindrical part of the AMV capsid favours the concept of dimers as the active assembling units.     

Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.     

Structure of the Top a-t component of alfalfa mosaic virus. A non-icosahedral virion

Neutron-scattering in combination with quasi-elastic light-scattering and electron microscopy was used to derive a model for the capsid structure of the Top a-t component of alfalfa mosaic virus (AMV-Ta-t). In the electron microscope, AMV-Ta-t appears as an irregular ellipsoidal particle with apparent dimensions 275 (+/- 31) A X 225 (+/- 22) A. Assuming that the particles are monodisperse, model calculations show that the neutron-scattering data are best explained by an oblate ellipsoidal shape for the virion with external dimensions 284 A X 284 A X 216 A. Based on this result, and in combination with the known composition of the virion, it is suggested that the capsid structure could be based on a deltahedron with 52 pointgroup symmetry and comprising 120 subunits. Such a model would imply a greater deviation from equivalent subunit interactions than normally necessary in icosahedral capsids. The neutron and photon correlation data, however, do not allow us to rule out the possibility that Top a-t is a slightly polydisperse preparation of irregular prolate shapes with mean dimensions 312 A X 232 A X 232 A. Both possibilities support the concept of alfalfa mosaic virus coat protein being capable of a wide range of intersubunit interactions, this flexibility resulting in considerable polymorphism in capsid structures.     

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.     

Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules

Structural changes in the single-stranded genome RNAs (RNAs 1, 2 and 3) and the subgenomic coat protein messenger (RNA 4) of alfalfa mosaic virus upon addition of a few coat protein molecules of the virus were investigated by measuring the fluorescent intensity of bound ethidium bromide and by circular dichroism. No effect could be observed in the case of the genome RNAs. However, in RNA 4, which is of much less complexity than the genome RNAs, a reduction of the ethidium bromide binding by 30% was found, whereas the positive molar ellipticity at 265 nm was reduced by 9% upon binding of the coat protein. Both changes point to a reduction of the ordered structure of the RNA. Since the protein is known to bind first at the 3′-terminus of RNA 4 and probably also of the genome RNAs, the conformational changes observed could be those thought to be necessary for replicase recognition in this positive-stranded RNA virus which needs the coat protein for starting an infection cycle.     

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.     

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.