Ribosome shunt is essential for infectivity of cauliflower mosaic virus

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicat ing by means of reverse transcription of a terminally redundant pregenomic 355 RNA that is also used as a polycistronic mRNA. The leader of 355 RNA is long, highly structured, and contains multiple short ORFs (sORFs), which s trongly interfere with the ribosome scanning process. Translation of this R NA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the l eader containing a putative RNA encapsidation signal and reinitiate transla tion at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of sh unt-dependent expression and the degree of shunt enhancement by a CaMV-enco ded translation transactivator (TAV), consequently reduced infectivity of t he virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shunt-dependent express ion to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shu nting proved to be lethal. Finally, by using an artificial stem structure t hat blocks scanning, we obtained direct evidence that ribosome shunt operat es during CaMV infection.