MUTATION ANALYSES OF MOLECULARLY CLONED SATELLITE TOBACCO MOSAIC-VIRUS DURING SERIAL PASSAGE IN PLANTS - EVIDENCE FOR HOTSPOTS OF GENETIC CHANGE

The high level of genetic diversity and rapid evolution of viral RNA g enomes are well documented, but few studies have characterized the rat e and nature of ongoing genetic change over time under controlled expe rimental conditions, especially in plant hosts. The RNA genome of sate llite tobacco mosaic virus (STMV) was used as an effective model for s uch studies because of advantageous features of its genome structure a nd because the extant genetic heterogeneity of STMV has been character ized previously. In the present study, the process of genetic change o ver time was studied by monitoring multiple serial passage lines of ST MV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcri bed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for ea ch line and the consensus genotypes of progeny STMV populations were a ssessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were obs erved, including the fixation of novel mutations in 9 of 42 lines, mut ation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major ge notype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substi tutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting ho t spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversific ation of STMV virus populations.