Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast
            <i>Pichia pastoris</i>

Hwang, Jung Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

doi:10.1128/jvi.74.9.4074-4084.2000

Cited by 20 publications

(14 citation statements)

References 47 publications

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“…Although NP is essential for in vivo replication of influenza virus RNA (2,14), the presence of NP was not required for the synthesis of short 14-nt transcripts (Fig. 1), confirming previous studies with short RNA templates (12,13,16,22). Recombinant RNA polymerase prepared by our method also required, as expected (7,8,13), both the 5Ј and 3Ј ends of the vRNA promoter for significant transcriptional activity (Fig.…”

Section: Discussionsupporting

confidence: 71%

The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter

Brownlee

Sharps

2002

J Virol

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The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation. We prepared partially purified recombinant influenza A virus RNA polymerase, in the absence of influenza virus vRNA promoter sequences, by transient transfection of expression plasmids into human kidney 293T cells. The polymerase was found to be heat labile at 40°C in the absence of added vRNA. However, it was protected from heat inactivation if both the 5 and 3 strands of the vRNA promoter were present. By using the ability of vRNA to protect the enzyme against heat inactivation, we established a novel assay, in conjunction with a mutagenic approach, that was used to test the secondary structure requirement of the vRNA promoter for polymerase binding. Binding required a panhandle structure and the presence of local hairpin loop structures in both the 5 and 3 ends of vRNA, as suggested by the corkscrew model. The interaction of the vRNA promoter with the influenza virus RNA polymerase heterotrimeric complex is likely to favor a particular closed conformation of the complex, thereby ensuring the stability of the RNA polymerase within both the infected cell and the isolated virus.Influenza virus is a negative-sense, segmented RNA virus with eight RNA segments coding for 10 genes. The three longest gene segments code for the three subunits PB1, PB2, and PA of the heterotrimeric influenza virus RNA polymerase complex, which is the enzyme responsible for transcription and replication of the viral genome in the infected cell. In addition to its role as an RNA-dependent RNA polymerase, which copies virion RNA (vRNA)3mRNA in transcription and vRNA3cRNA and cRNA3vRNA in replication, the complex is also an endonuclease, snatching capped RNAs from host cell pre-mRNA for use as primers for the synthesis of influenza virus-specific mRNA, and a poly(A) polymerase, catalyzing polyadenylation of influenza virus-specific mRNA (for reviews, see references 19, 24, and 31).There has been significant progress in understanding the sequence and secondary structure properties of the vRNA promoter, which consists of 13 nucleotides (nt) at the 5Ј end and 12 nt at the 3Ј end of the vRNA, all of which are conserved in each RNA segment of every influenza A virus. These conserved nucleotides, along with an extra one to five segmentspecific bases at the 5Ј and 3Ј ends of vRNA, form a partially double-stranded panhandle structure. Additional short hairpin loops, consisting of a 2-bp stem and a tetraloop, have been proposed either at the 5Ј end (5Ј hairpin loop model) or at both the 5Ј and 3Ј ends (corkscrew model) of the vRNA (4, 33).The hairpin loop near the 5Ј end of the vRNA pr...

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Section: Discussionsupporting

confidence: 71%

The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter

Brownlee

Sharps

2002

J Virol

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“…3C). A viral polymerase forms a stable complex (1,36,45), and we confirmed that the N-terminal epitopetagged polymerase subunits used in this study are also able to form a complex (Fig. 1).…”

Section: Inhibition Of Ips-1-mediated Induction Of Type I Ifn Throughsupporting

confidence: 66%

Influenza A Virus Polymerase Inhibits Type I Interferon Induction by Binding to Interferon β Promoter Stimulator 1

Iwai

Shiozaki

Kawai

et al. 2010

Journal of Biological Chemistry

112

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Type I interferons (IFNs) are known to be critical factors in the activation of host antiviral responses and are also important in protection from influenza A virus infection. Especially, the RIG-I-and IPS-1-mediated intracellular type I IFN-inducing pathway is essential in the activation of antiviral responses in cells infected by influenza A virus. Previously, it has been reported that influenza A virus NS1 is involved in the inhibition of this pathway. We show in this report that the influenza A virus utilizes another critical inhibitory mechanism in this pathway. In fact, the viral polymerase complex exhibited an inhibitory activity on IFN␤ promoter activation mediated by RIG-I and IPS-1, and this activity was not competitive with the function of NS1. Co-immunoprecipitation analysis revealed that each polymerase subunit bound to IPS-1 in mammalian cells, and each subunit inhibited the activation of IFN␤ promoter by IPS-1 independently. In addition, by a combinational expression of each polymerase subunit, IPS-1-induced activation of IFN␤ promoter was more efficiently inhibited by the expression of PB2 or PB2-containing complex. Moreover, the expression of PB2 inhibited the transcription of the endogenous IFN␤ gene induced after influenza A virus infection. These findings demonstrate that the viral polymerase plays an important role for regulating host anti-viral response through the binding to IPS-1 and inhibition of IFN␤ production.

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“…4). The activity of model vRNA-directed and primer-dependent RNA synthesis by the 3P complex was the highest among those obtained by various methods so far established in this laboratory (18,23,38,41).…”

Section: Discussionmentioning

confidence: 75%

“…To overcome the above failures to obtain large amounts of the functional RNA polymerase in RNA-free form, we have developed an expression system of functional form of influenza virus RNA polymerase in methylotrophic yeast Pichia pastoris (41), but the purification of RNA polymerase without contamination of endogenous nucleases was difficult. Here we succeeded in expressing all three P proteins simultaneously in insect cells after coinfection with three species of the recombinant baculovirus, each coding for PB1, PB2, and H-PA.…”

Section: Discussionmentioning

confidence: 99%

Differential Roles of Viral RNA and cRNA in Functional Modulation of the Influenza Virus RNA Polymerase

Honda¹,

Endo²,

Mizumoto³

et al. 2001

Journal of Biological Chemistry

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The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNAdirected RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirusinfected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.The genome of influenza virus is composed of eight negativestrand viral RNA (vRNA) 1 segments, which altogether encode 10 different viral proteins (1). The genome RNAs are transcribed in virus-infected cell nuclei (2, 3) into mRNA by the virion-associated RNA-dependent RNA polymerase (4). Genetic and biochemical studies indicate that this RNA polymerase is involved in both transcription (vRNA-directed mRNA synthesis) and replication (vRNA-directed synthesis of complementary RNA (cRNA) and cRNA-directed vRNA synthesis) (5, 6). In transcription, the RNA polymerase catalyzes not only viral RNA synthesis but also the cleavage of capped host cell RNA to generate capped RNA primers for viral mRNA synthesis (7-11) and polyadenylation at the 3Ј-termini of mRNA (12-15). The RNA polymerase also carries an apparent proofreading activity for nascent RNA chains (16).The RNA polymerase is composed of one molecule each of three viral proteins, PB1, PB2, and PA (17, 18). The PB1 protein plays a central role in RNA polymerase assembly by providing the contact surfaces for both PB2 and PA (19). In the virus particles, this RNA polymerase is bound at a doublestranded region of vRNA formed by base pairing between its 5Ј-and 3Ј-termini (20, 21). Since the RNA polymerase is tightly associated with vRNA (18) and since the content of RNA polymerase in virions is very low, i.e. less than 1% of total virion proteins (1, 22), no purification method yielding large amounts of the functional RNA polymerase has been established. Previously Kobayashi et al. (23) demonstrated that the viral RNA polymerase with RNA synthesis activity could be reco...

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Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Cited by 20 publications

References 47 publications

The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter

The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter

Influenza A Virus Polymerase Inhibits Type I Interferon Induction by Binding to Interferon β Promoter Stimulator 1

Differential Roles of Viral RNA and cRNA in Functional Modulation of the Influenza Virus RNA Polymerase

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