Unstable infectivity and sedimentable ds-RNA associated with lettuce speckles mottle virus

Infectivity associated with extracts of lettuce speckles mottle virus (LSMV)-infected tissues is unstable and is susceptible to short periods of aging in vitro or low concentrations of RNase. Infected leaves extracted using phenol yielded greater infectivity than comparable tissue extracted with buffer. No typical virus particles were observed in infected tissues or clarified preparations, but 50- to 70-nm spherical membranous particles were observed associated with the tonoplast in vacuoles of infected cells. Polyacrylamide gel electrophoresis of nucleic acids from infected tissue and LSMV-clarified preparations revealed abundant double-stranded RNA (ds-RNA) in both preparations. A species of LSMV-specific single-stranded RNA (ss-RNA) was also present in LSMV bentonite-clarified preparations. Infectivity was associated only with the ss-RNA fraction. Quantitative isolation of ds-RNA from whole tissue and clarified preparations showed the majority of the ds-RNA was recovered from the bentonite-clarified fractions, and this RNA appeared to be associated with a relatively small structure of a low buoyant density. These data suggest that LSMV lacks a functional coat protein and the sedimentable dsRNA fraction from LSMV-infected tissues more likely contains a viral-specific replication site and not true virus particles.