Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo.

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.

Host-controlled cell-to-cell movement of a hybrid barley stripe mosaic virus expressing a dianthovirus movement protein.

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.

Movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein.

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNA beta replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs alpha and gamma, together with the hybrid RNA beta transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves of Nicotiana benthamiana and Chenopodium amaranticolor, which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions on C. amaranticolor were influenced by the foreign MP gene. However, the hybrid virus failed to infect barley, N. tabacum (var. Samsun), and N. clevelandii, the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Movement of hordeivirus hybrids with exchanges in the triple gene block.

The barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infected Nicotiana benthamiana, a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (beta b gene) indicated that (i) sequence-independent binding of beta b to viral RNAs could occur during formation of beta b-RNA complexes in vivo, and that (ii) the beta b MP is involved in virus long-distance movement, for which homologous N- and C-terminal beta b domains are required.