Replication of alfalfa mosaic virus RNA: evidence for a soluble replicase in healthy and infected tobacco leaves.
Soluble RNA-dependent RNA polymerases from healthy and alfalfa mosaic virus (AMV)-infected leaves were purified more than 400-fold from the 100,000g supernatant of leaf homogenates, using ammonium sulfate precipitation, Sephadex G-100 filtration, and chromatography on DEAE Sephadex A25 and P11-phosphocellulose. The DEAE-chromatography step eliminated host poly(U)polymerase-like activity, cellular RNases, and endogenous RNA, yielding a typical RNA-dependent RNA polymerase ("DEAE-enzyme") which was dependent on exogenous RNA. On nondenaturing 4-30% gradient slab gels, the activity of the enzyme was recovered in two peaks containing proteins of 340K and 160K. RNA products were synthesized in the presence of the "DEAE-enzyme" from healthy and infected leaves: When an excess of AMV RNA was introduced into the reaction mixture, only synthesis of minus strand RNA was detected. However, when limiting amounts of AMV RNA were used as template some small plus strands were detected, suggesting that synthesized minus strands may in turn serve as template. In both cases, the products were partially double stranded, and of very heterogenous size. Their size depended on the length of the RNA template which in turn varied according to the degree of RNase contamination of the enzyme fractions. These results are the first demonstration that both healthy and AMV-infected tobacco leaves contain a soluble replicase, although its possible in vivo role in viral replication is yet to be demonstrated.
Chifflot
S
S
Laboratoire de Virologie, Institut de Biologie Mole culaire et Cellulaire du CNRS, 15 rue Descartes, 67000 Strasbourg, France.
Sommer
P
P
Hartmann
D
D
Stussi-Garaud
C
C
Hirth
L
L