-
[show abstract]
[hide abstract]
ABSTRACT: The cap-dependent endonuclease activity of the influenza virus RNA-dependent RNA polymerase cleaves host mRNAs to produce capped RNA fragments for primers to initiate viral mRNA synthesis. The influenza A virus (FluA) cap-dependent endonuclease preferentially recognizes the cap1 structure (m(7)GpppNm). However, little is known about the substrate specificity of the influenza B virus (FluB) endonuclease. Here, we determined the substrate specificity of the FluB polymerase using purified viral RNPs and (32)P-labeled polyribonucleotides containing a variety of cap structures (m(7)GpppGm, m(7)GpppG, and GpppG). We found that the FluA polymerase cleaves m(7)G-capped RNAs preferentially. In contrast, the FluB polymerase could efficiently cleave not only m(7)G-capped RNAs but also unmethylated GpppG-RNAs. To identify a key amino acid(s) related to the cap recognition specificity of the PB2 subunit, the transcription activity of FluB polymerases containing mutated cap-binding domains was examined by use of a minireplicon assay system. In the case of FluA PB2, Phe323, His357, and Phe404, which stack the m(7)GTP, and Glu361 and Lys376, which make hydrogen bonds with a guanine base, were essential for the transcription activity. In contrast, in the case of FluB PB2, the stacking interaction of Trp359 with a guanine base and putative hydrogen bonds using Gln325 and Glu363 were enough for the transcription activity. Taking these results together with the result for the cap-binding activity, we propose that the cap recognition pocket of FluB PB2 does not have the specificity for m(7)G-cap structures and thus is more flexible to accept various cap structures than FluA PB2.
Journal of Virology 05/2011; 85(15):7504-12. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.
Journal of Biological Chemistry 03/2005; 280(6):4429-35. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cellular tubulin has been shown to activate in vitro transcription with Sendai virus (SeV) particles. In this study, the molecular basis for the transcriptional activation by tubulin was investigated. We showed that tubulin dissociates viral matrix (M) protein, which acts as a negative regulator for transcription, from viral ribonucleoprotein (RNP) consisting of L, P, N proteins, and the genome RNA. Both alpha and beta subunits of human tubulin, which were expressed as GST fusion proteins, were found to stimulate viral mRNA synthesis similar to native alpha/beta-heterodimer tubulin. Pull-down assay using GST-tubulin subunits demonstrated that M protein is released from the RNP as a complex with each tubulin subunit. In vitro-binding analyses revealed that M protein directly interacts with tubulin as well as microtubules. These findings suggest that interaction of M protein with tubulin may have an important role in the regulation of SeV transcription.
Biochemical and Biophysical Research Communications 12/2003; 311(2):283-93. · 2.48 Impact Factor
