Non-structural proteins and RNAs of alfalfa mosaic virus synthesized in tobacco and cowpea protoplasts.

Three proteins, reacting specifically with sera raised against synthetic peptides identical to C-terminal amino acid sequences in alfalfa mosaic virus (AIMV) proteins P1, P2, and P3 translated in vitro from the AIMV RNAs 1, 2, and 3, respectively, were for the first time observed in tobacco and cowpea protoplasts. Part of P2 is post-translationally modified in protoplasts, because the anti-P2 serum reacted also with a protein migrating slower than P2 itself. The modification reported for P3 in infected tobacco leaves (T. Godefroy-Colburn et al. (1986) J. Gen. Virol. 67, 2233-2241) was observed in AIMV-infected bean leaves but not in AIMV-infected protoplasts and is apparently not essential for viral replication. Time course experiments showed that all nonstructural proteins could be detected 6 hr postinoculation. The two largest proteins P1 and P2 disappeared when virus production had reached a plateau, while the smallest nonstructural protein P3 remained at a constant level. Cell fractionation experiments showed that minus-strand RNA as well as all viral-encoded proteins were found in the 1000 g subcellular fraction. This location differs from the location of the nonstructural proteins in infected tobacco leaves (A. Berna et al. (1986) J. Gen. Virol. 67, 1135-1147).

Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts.

Four proteins unique to virus infection were synthesized in alfalfa mosaic virus-infected alfalfa mesophyll protoplasts. These proteins, P1, P2, P3, and coat protein comigrated on electrophoresis with the major in vitro translation products of RNA 1, RNA 2, RNA 3, and RNA 4, respectively. P1, P3, and coat protein were observed at 5 hr post inoculation; P2 was detected at 9 hr post inoculation. The three nonstructural proteins accumulated most rapidly early in infection until about 15 hr post inoculation; stable protein levels were maintained thereafter. Coat protein accumulated rapidly until about 20 hr after inoculation. All four virus RNA species were detected in infected protoplasts by labelling with [3H]uridine. Ultraviolet irradiation of protoplasts prior to inoculation was necessary for virus protein detection, but it severely depressed the synthesis of RNA 1 and RNA 2 relative to RNA 3 and RNA 4.