Influence of N and NSs on translation.

<p>BHK-21 cells were infected with <i>Vaccinia virus</i> and co-transfected with 100 ng of expression vectors encoding REN luciferase, FF luciferase and MBP, N, NSs, combination of N and NSs, or pUC19 at the amount of 450 ng (A) and 700 ng (B). pUC19 was added as negative control. Luciferase expression was measured 23 h post transfection. The relative luciferase expression is shown, corrected for the internal FF control (REN/FF). (C) Cells were analysed for expression of MBP, N, or NSs by Western blotting and using antisera specific for MBP, N or NSs respectively. Abbreviation: MBP, Maltose binding protein; N, nucleoprotein; NSs, non-structural protein; H, hairpin; ½H, half hairpin; pA, polyA; (-), negative control.</p

Analysis of the A- and U-rich part of the predicted hairpin structure sequence in translation.

<p>(A) Localization of the A- and U-rich part within the predicted hairpin structure sequence. (B) BHK-21 cells were infected with vv-T7 and co-transfected with 100 ng of pREN sensor constructs (pREN-H^A/U-rich, pREN-halfH^A-rich, pREN-halfH^A*-rich, pREN-halfH^U-rich, or pREN-polyA) and 0.5 ng of pIRES-FF as internal control. Relative luciferase expression was measured after 23 h post transfection. Error bars show the standard deviations from the means of three replicate experiments.</p

Structural features within the S RNA segment.

<p>Structural features within the S RNA segment.</p

Requirement of the 5′ UTR sequence in translation.

<p>BHK-21 cells were infected with <i>Vaccinia virus</i>, and subsequently co-transfected with 100 ng of the indicated REN constructs and 0.5 ng of pIRES-FF as internal control. The relative luciferase expression (REN/FF) was measured after 23 h post transfection. Error bars show the standard deviations from the means of three replicate experiments.</p

Comparison of the predicted hairpin structure sequence from TSWV (N gene transcript) with the analogous one from TYRV.

<p>(A) Alignment of the TSWV and TYRV N-based hairpin structure sequence. (B) Predicted hairpin structure at the 3′ end of the N gene of TSWV and TYRV respectively. (C) BHK-21 cells were infected with <i>Vaccinia virus</i> and transfected with 100 ng of either pREN-H^A/U-rich, pREN-TYRV H, or pREN-polyA. In addition to the REN construct, 0.5 ng of pIRES-FF was added as internal control. After 23 h, the cells were lysed and assayed for relative luciferase activity. Error bars show the standard deviations from the means of three replicate experiments.</p