How can our results be reconciled with previous results which indicated that only the immediately 3 0 -adjacent 1-3 nt were relevant for RT in these viruses? There are several possibilities. Previous analyses of the RT cassette in SINV alphavirus, and also tobacco rattle tobravirus, were performed in in vitro systems (32, 33) . However, RT efficiency may vary considerably between in vitro and cell culture systems, depending on the absence or presence and abundance of various relevant near-cognate tRNA species (34) , and potentially also on the concentration of various trans-acting factors, salt concentrations, temperature, ribosome loading density and intracellular architecture. Thus a high RT efficiency measured in vitro for a short insert does not mean that the full complement of elements that stimulate efficient RT in cell culture or in vivo has been recapitulated faithfully. Such factors may also explain why our in vitro experiments produced much lower RT efficiencies than previous in vitro experiments, and highlight the importance of our experiments in mammalian cell culture (32, 76, 77) . Although ref. (32) compared, in vitro, the RT efficiency for a short insert (that excluded the predicted structure) with a long insert (comprising the entire nsP3+nsP4-coding sequences), such comparisons between inserts of very different sizes are not always straightforward, in part because the different protein products may be degraded at different rates, and because chance base pairings with the construct sequence could affect RT efficiency differently for the long and short inserts. In contrast, guided by our computational analysis, we were able to make small but targeted substitutions that allowed for more precise comparisons in the context of a long VEEV insert that included the predicted RNA structure elements. Accurate measurements of the RT efficiency in alphavirus-infected cells are not readily obtainable due to the multiple cleavage products of the non-structural polyprotein and rapid degradation of excess nsP4 (31, 47, 78) . Nonetheless, in ref. (31) , 5-to 8-fold less nsP34 was found in WT SINV-infected cells than in cells infected with mutant viruses in which the UGA was replaced by a Ser, Trp or Arg codon, thus suggesting a WT RT efficiency in the range 12.5-20%. Our measurement of $7% for WT VEEV and SINV sequences in the dual luciferase construct suggests that there may be additional factors that affect alphavirus RT.