Genome transcription/translation of segmented, negative-strand RNA viruses

The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a preference for leaders with increasing base complementarity to the 3'-ultimate residues of the viral RNA template. Assuming that cap-snatching is a highly conserved mechanism, it is tempting to speculate that the findings for TSWV apply to all segmented negative RNA viruses. The research in this thesis aimed to analyze whether similar cap donor requirements applied for Influenza A virus transcription initiation as compared to what has been found for TSWV. Indeed, in vitro studies demonstrated that influenza transcriptase prefers multiple base-pairing capped leaders. Additionally, the occurrence of “prime-and-realign” during influenza transcription initiation was observed, as well as internal priming at the 3'-penultimate viral residue. The in vitro findings were confirmed by similar studies performed during influenza infection of cell cultures. Whereas transcription initiation of TSWV has been relatively well studied, transcription termination has not. It is postulated that transcription termination/translation is triggered by the formation of a hairpin structure. In cell experiments support a role of the TSWV hairpin structure in translation.</p

Non-structural proteins of arthropod-borne bunyaviruses: roles and functions

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod

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

Preferential use of RNA leader sequences during influenza A transcription initiation in vivo

In vitro transcription initiation studies revealed a preference of influenza A virus for capped RNA leader sequences with base complementarity to the viral RNA template. Here, these results were verified during an influenza infection in MDCK cells. Alfalfa mosaic virus RNA3 leader sequences mutated in their base complementarity to the viral template, or the nucleotides 5' of potential base-pairing residues, were tested for their use either singly or in competition. These analyses revealed that influenza transcriptase is able to use leaders from an exogenous mRNA source with a preference for leaders harboring base complementarity to the 3'-ultimate residues of the viral template, as previously observed during in vitro studies. Internal priming at the 3'-penultimate residue, as well as “prime-and-realign” was observed. The finding that multiple base-pairing promotes cap donor selection in vivo, and the earlier observed competitiveness of such molecules in vitro, offers new possibilities for antiviral drug design

Base-pairing promotes leader selection to prime in vitro influenza genome transcription

AbstractThe requirements for alignment of capped leader sequences along the viral genome during influenza transcription initiation (cap-snatching) have long been an enigma. In this study, competition experiments using an in vitro transcription assay revealed that influenza virus transcriptase prefers leader sequences with base complementarity to the 3′-ultimate residues of the viral template, 10 or 11 nt from the 5′ cap. Internal priming at the 3′-penultimate residue, as well as prime-and-realign was observed. The nucleotide identity immediately 5′ of the base-pairing residues also affected cap donor usage. Application to the in vitro system of RNA molecules with increased base complementarity to the viral RNA template showed stronger reduction of globin RNA leader initiated influenza transcription compared to those with a single base-pairing possibility. Altogether the results indicated an optimal cap donor consensus sequence of 7mG-(N)7–8-(A/U/G)-(A/U)-AGC-3′

Analysis of the hairpin structure sequence in translation.

<p>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.</p

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

<p>(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.</p

RNA folding predictions of TSWV M segment.

<p>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.</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

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