Analysis of the Tomato spotted wilt virus ambisense S RNA-encoded hairpin structure in translation

Abstract

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 3' 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 3' and/or 5' 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 5' UTR and the 3' UTR, luciferase expression was lost when the hairpin structure sequence was removed from the 3' UTR. Constructs that only lacked the 5' UTR, still rendered good expression levels. When in addition the entire 3' 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 5' 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 luciferase expression levels from hairpin structure containing constructs.

Conclusions/significance: The results suggest a role for the predicted hairpin structure in translation in concert with the viral N and NSs proteins. The presence of stretches highly rich in A-residues does not rule out a concerted action with a poly(A)-tail-binding protein. A common transcription termination and translation strategy for plant- and animal-infecting ambisense RNA viruses is being discussed.

Figure 1. Structural features within the S RNA segment.

Figure 2. Analysis of the hairpin structure sequence in translation.

Schematic presentation of TSWV-N (REN) and derived templates with modifications at the 3′ UTR (A and B). (C) Luciferase activity monitored from REN constructs transfected to BHK-21 cells. Cells were infected with vv-T7 and subsequently co-transfected with 100 ng of the indicated REN constructs and 0.5 ng of the FF luciferase expression plasmid (pIRES-FF) as internal control. The relative luciferase expression (REN/FF) was measured after 23 h post transfection. Error bars indicate standard deviations from the means of three replicate experiments.

Figure 3. Requirement of the 3′ UTR of TSWV mRNAs in translation.

(A) Sequence alignment of the TSWV N gene 3′ UTR (pREN-H^A/U-rich) and its reverse complement (pREN-H^A/U*-rich). (B) Mfold predictions of the highly AU-rich sequence in the viral sense RNA (vRNA) flanking the 3′ end of the NSs ORF (pREN-H^A/U*-rich, panel A), and the analogous sequence in the viral complementary RNA (vcRNA) flanking the 3′ end of the N ORF (pREN-H^A/U-rich, panel A). (C) Luciferase activity measured from BHK-21 cells infected with vv-T7 and subsequently co-transfected with 100 ng of expression REN constructs (pREN-H^A/U-rich, pREN-H^A/U*-rich, or pREN-polyA) 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.

Figure 4. Requirement of the 5′ UTR sequence in translation.

BHK-21 cells were infected with Vaccinia virus, 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.

Figure 5. Comparison of the predicted hairpin structure sequence from TSWV (N gene transcript) with the analogous one from TYRV.

(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 Vaccinia virus 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.

Figure 6. Analysis of the A- and U-rich part of the predicted hairpin structure sequence in translation.

(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.

Figure 7. Influence of N and NSs on translation.

BHK-21 cells were infected with Vaccinia virus 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.

Figure 8. RNA folding predictions of TSWV M segment.

Mfold predictions of the highly AU-rich IR in the vcRNA flanking the 3′ end of the G precursor ORF (A), and the analogous sequence in the vRNA flanking the 3′ end of the NSm ORF (B), . Abbreviation: vRNA, viral sense RNA; vcRNA, viral complementary RNA.