Preferential use of RNA leader sequences during influenza A transcription initiation in vivo
Geerts-Dimitriadou, C. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2011
Virology 409 (2011)1. - ISSN 0042-6822 - p. 27 - 32.
b-virus genome - complete nucleotide-sequence - cap-snatching mechanism - open reading frame - messenger-rna - viral-rna - 5' ends - base complementarity - neuraminidase gene - alpha-amanitin
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
Geerts-Dimitriadou, C. ; Zwart, M.P. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2011
Virology 409 (2011)1. - ISSN 0042-6822 - p. 17 - 26.
messenger-rna synthesis - cap-snatching mechanism - mosaic-virus rnas - viral-rna - 5' ends - complementary rna - a virus - polymerase - endonuclease - sequences
The 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'.
Tomato spotted wilt virus transcriptase in vitro displays a preference for cap donors with multiple base complementary to the viral template
Knippenberg, I.C. van; Lamine, M. ; Goldbach, R.W. ; Kormelink, R.J.M. - \ 2005
Virology 335 (2005)1. - ISSN 0042-6822 - p. 122 - 130.
messenger-rna synthesis - influenza-virus - 5' ends - mosaic-virus - independent translation - heterogeneous sequences - nsm protein - s-rna - polymerase - bunyavirus
Transcription of segmented negative-strand RNA viruses is initiated by cap snatching: a host mRNA is cleaved generally at 10¿20 nt from its 5¿ capped end and the resulting capped leader used to prime viral transcription. For Tomato spotted wilt virus (TSWV), type species of the plant-infecting Tospovirus genus within the Bunyaviridae, cap donors were previously shown to require a single base complementarity to the ultimate or penultimate viral template sequence. More recently, the occurrence in vitro of ¿re-snatching¿ of viral mRNAs, i.e., the use of viral mRNAs as cap donors, has been demonstrated for TSWV. To estimate the relative occurrence of re-snatching compared to snatching of host mRNAs, the use of cap donors with either single, double, or multiple complementarity to the viral template was analyzed in pair-wise competition in TSWV in vitro transcription assays. A strong preference was observed for multiple-basepairing donors