Competitive Multiplication of RNA3 Species of Different Strains of Alfalfa Mosaic Virus

Abstract

SUMMARYCompetition between RNA3 from alfalfa mosaic virus (AIMV) strain S (RNA3-S), strain B (RNA3-B) and strain 425L (RNA3-L) was studied. The identification of the RNA3 species multiplying in infected leaves was possible since the RNA3 5' noncoding leader sequences in strains S, B and 425L differ in length. RNA3 present in total RNA from infected tobacco leaves was detected, and strains were identified from the length of the cDNA reverse-transcribed from RNA primed with a specific oligonucleotide. In competition experiments the inoculum, containing known amounts of RNA1, 2, 3 and 4 of one strain, was complemented with various amounts of heterologous RNA3 and inoculated to a systemic host. It is shown that RNA3-S was better replicated in vivo by the A1MV replicase of strain B than was RNA3-B itself, and to a lesser extent better replicated by the AIMV replicase of strain L than was RNA3-L. Comparison of genetic information carried by the RNA3 species present in the inoculum suggests that the more efficient multiplication of RNA3-S is related to the structure of the leader sequence of RNA3-S.The genome of alfalfa mosaic virus (A1MV) consists of three single-stranded RNA species: RNA1, RNA2 and RNA3. RNA1 and 2 encode proteins implicated in viral RNA replication (Nassuth & Bol, 1983) and RNA3, a bicistronic RNA, codes for a non-structural protein (P3) thought to be involved in cell-to-cell spread (Godefroy-Colburn et al., 1986) and for the viral coat protein (P4). The coat protein is expressed from a subgenomic RNA4. Genomic RNAs and RNA4 (or its translation product) are needed to initiate infection of plants (Bol et al., 1971).The four viral RNAs are separately encapsidated in four distinct particles (Bol et al., 1971) and their relative amounts in virus preparations depend on the strain of virus when multiplication is carried out in the same host under standard conditions. In the three strains used here, A1MV-425 isolate L (A1MV-L) (Bol et al., 1971), A1MV-S and AIMV-B (Walter et al., 1985), the relative proportions of viral RNA were as shown in Fig. 1. There was a clear difference between the three strains in the amounts of RNA3 and RNA4; in particles of strain L there was little RNA3 but much RNA4, this ratio was reversed in strain S, and in particles of strain B both RNAs were equally abundant as judged by the intensity of staining with toluidine blue.The lengths of the leader sequences of RNA3 of the three strains of A1MV, i.e. the sequences from the 5' end to the first AUG initiation codon, were 240 nucleotides for strain B (Ravelonandro et al., 1983), 314 nucleotides for strain S and 345 nucleotides for strain 425L (Langereis et al., 1986). These leader sequences show characteristic features: two stretches of 56 and 76 nucleotides are duplicated in RNA3-S and RNA3-L respectively, a 27 to 30 nucleotide region is repeated four times in RNA3-S and RNA3-L and three times in RNA3-B, and the RNA3-L contains an A-rich region of 40 nucleotides (Langereis et al., 1986; Dore & Pinck, 1988). Based on the RNA3 seque...

Biologically active transcripts of alfalfa mosaic virus RNA3

“…They will be used to study the molecular aspects of the virus multiplication. As previously reported, the amount of RNA3 relative to that of RNA4 extracted from the virions of strains S, B, and 425 L differed [24], and furthermore this ratio seems not to be influenced by the strain from which RNA1 and RNA2 originated (to be published).…”

“…This treatment enhanced the amount of RNA3-S transcripts produced by the T7 polymerase and prevented the synthesis of RNA products of larger 184 Capped transcripts were used to complement a mixture of RNA1,2 of strain B and coat protein of strain 425L. RNA 1 and 2 were purified from strain B of AIMV and coat protein from strain 425L because it is easy to distinguish RNA3 molecules of strain S, B and 425L by primer extension [24] since the length of their leader is different ([4,5] and unpublished results). An oligodeoxynucleotide hybridizing specifically on all RNA3s in the P3 open reading frame near the AUG initiation codon was used as primer to synthesize fulllength cDNA on RNA3 present in total RNA extracted from leaves.…”

“…Then the length of the cDNA was analyzed in a sequencing gel to identify the strain origin of the RNA3 reverse transcribed. The full-length cDNA is in increasing order RNA3-B, RNA3-S, RNA3-L [24].…”

“…The inoculation was done with 20~1 of a mixture containing 1.6,ug RNAl, 1.2 pg RNA2 prepared from the AlMV B strain as previously described [24] and coat protein from strain L prepared according to [25]. Young tobacco plants with three leaves (2-3 cm width) were used.…”

“…RNAs were extracted from the inoculated leaf 6 days after inoculation and from a systemic upper leaf 8 days after inoculation as indicated in [26]. RNA3 detection and strain characterization by primer extensions were done as in [24].…”