Alfalfa mosaic virus temperature-sensitive mutants. II. Early functions encoded by RNAs 1 and 2.

Mutants Bts 03 and Mts 04 of alfalfa mosaic virus (AIMV) have temperature-sensitive mutations in genomic RNAs 1 and 2, respectively. These mutants are defective in the production of viral minus-strand RNA, coat protein, and infectious virus when assayed in cowpea protoplasts at the nonpermissive temperature (30 degrees). To determine the temperature-sensitive step in the replication cycle, mutant-infected protoplasts were shifted from an incubation temperature of 25 degrees (permissive temperature) to 30 degrees at different times during a 24-hr incubation period.

Inhibition of alfalfa mosaic virus RNA and protein synthesis by actinomycin D and cycloheximide.

Actinomycin D, added early after inoculation, reduces the production of infectious alfalfa mosaic virus in cowpea protoplasts by 90%. This reduction was associated with an inhibition of viral minus-strand and plus-strand RNA synthesis, suggesting the involvement of host factors in these processes. Coat protein production was less affected by the drug.

An alfalfa mosaic virus RNA 2 mutant, which does not induce a hypersensitive reaction in cowpea plants, is multiplied to a high concentration in cowpea protoplasts.

A mutant of alfalfa mosaic virus (AMV), which in contrast to wild type (wt) can invade cowpea plants systemically, is replicated more efficiently in cowpea protoplasts than the wt. Mutant preparations isolated from infected cowpea protoplasts contained a higher amount of middle component (M, containing RNA 2) than wt preparations. Both in cowpea plants and in cowpea protoplasts a wt phenotype is obtained upon addition of wt M to this mutant, suggesting a correlation between the type of plant reaction evoked by the virus infection and the regulation of viral RNA synthesis.