Analysis of the Tomato spotted wilt virus Ambisense S RNA-Encoded Hairpin Structure in Translation

Background: The intergenic region (IR) of ambisense RNA segments from animal- and plant-infecting (-)RNA viruses functions as a bidirectional transcription terminator. The IR sequence of the Tomato spotted wilt virus (TSWV) ambisense S RNA contains stretches that are highly rich in A-residues and U-residues and is predicted to fold into a stable hairpin structure. The presence of this hairpin structure sequence in the 39 untranslated region (UTR) of TSWV mRNAs implies a possible role in translation. Methodology/Principal Findings: To analyse the role of the predicted hairpin structure in translation, various Renilla luciferase constructs containing modified 39 and/or 59 UTR sequences of the TSWV S RNA encoded nucleocapsid (N) gene were analyzed for expression. While good luciferase expression levels were obtained from constructs containing the 59 UTR and the 39 UTR, luciferase expression was lost when the hairpin structure sequence was removed from the 39 UTR. Constructs that only lacked the 59 UTR, still rendered good expression levels. When in addition the entire 39 UTR was exchanged for that of the S RNA encoded non-structural (NSs) gene transcript, containing the complementary hairpin folding sequence, the loss of luciferase expression could only be recovered by providing the 59 UTR sequence of the NSs transcript. Luciferase activity remained unaltered when the hairpin structure sequence was swapped for the analogous one from Tomato yellow ring virus, another distinct tospovirus. The addition of N and NSs proteins further increased luciferas

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

Structural features within the S RNA segment.

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

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