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

ABSTRACT Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoriswas achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.

Download Full-text

Mesoionic Heterocyclic Compounds as Candidate Messenger RNA Cap Analogue Inhibitors of the Influenza Virus RNA Polymerase Cap-Binding Activity

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020901900504 ◽

2009 ◽

Vol 19 (5) ◽

pp. 213-218 ◽

Cited By ~ 6

Author(s):

Ian Mickleburgh ◽

Feng Geng ◽

Laurence Tiley

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Messenger Rna ◽

Binding Activity ◽

Endonucleolytic Cleavage ◽

Virus Rna ◽

Fused Ring ◽

Mesoionic Heterocycles ◽

Potential Inhibitors

Background: An unusual feature of influenza viral messenger RNA (mRNA) synthesis is its dependence upon host cell mRNAs as a source of capped RNA primers. A crucial activity of the influenza polymerase is to steal these primers by binding and cleaving the caps from host mRNAs. The recent structural analysis of the cap-binding fragment of the influenza virus PB2 protein has highlighted the importance of the mesoionic properties of the N7-methylguanine (N7mG) component of the mRNA cap in this interaction. Methods: A series of mesoionic heterocycles with 5,6-fused ring systems analogous to the N7mG component of mRNA cap structures were synthesized and examined for the ability to inhibit the cap-binding activity of the influenza virus RNA polymerase complex using a bead-based in vitro cap-binding assay. Results: None of the compounds tested were able to significantly inhibit binding and subsequent endonucleolytic cleavage of a synthetic radiolabelled capped mRNA substrate by recombinant influenza virus polymerase in vitro. Conclusions: Compounds analogous to the mesoionic N7mG component of mRNA cap structures comprise a large class of potential inhibitors of the influenza virus polymerase. Although this preliminary assessment of a small group of related analogues was unsuccessful, further screening of this class of compounds is warranted.

Download Full-text

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02605-20 ◽

2021 ◽

Vol 65 (4) ◽

Author(s):

Aartjan J. W. te Velthuis ◽

Tatiana G. Zubkova ◽

Megan Shaw ◽

Andrew Mehle ◽

David Boltz ◽

...

Keyword(s):

Influenza Virus ◽

Placebo Group ◽

Rna Polymerase ◽

Rna Synthesis ◽

Respiratory Infections ◽

Respiratory Viruses ◽

Virus Shedding ◽

Virus Rna ◽

Viral Respiratory Infections ◽

Viral Respiratory

ABSTRACT Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.)

Download Full-text

Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Journal of Virology ◽

10.1128/jvi.72.8.6546-6553.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6546-6553 ◽

Cited By ~ 64

Author(s):

Julie A. Lemm ◽

Anders Bergqvist ◽

Carol M. Read ◽

Charles M. Rice

Keyword(s):

Rna Polymerase ◽

Sindbis Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Functional Assay ◽

Minus Strand ◽

Virus Rna ◽

A Genome ◽

Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Download Full-text

Enisamium Inhibits SARS-CoV-2 RNA Synthesis

Biomedicines ◽

10.3390/biomedicines9091254 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1254

Author(s):

Stefano Elli ◽

Denisa Bojkova ◽

Marco Bechtel ◽

Thomas Vial ◽

David Boltz ◽

...

Keyword(s):

Cell Culture ◽

Rna Polymerase ◽

Influenza A ◽

Rna Synthesis ◽

Herd Immunity ◽

Virus Rna ◽

Independent States ◽

Dynamics Simulations ◽

Insight Into

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.

Download Full-text

Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/30.2.429 ◽

2002 ◽

Vol 30 (2) ◽

pp. 429-438 ◽

Cited By ~ 60

Author(s):

M. T. M. Lee

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Rna Synthesis ◽

Virus Rna ◽

Definition Of ◽

Viral Components

Download Full-text

Mechanism of Action of T-705 against Influenza Virus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.3.981-986.2005 ◽

2005 ◽

Vol 49 (3) ◽

pp. 981-986 ◽

Cited By ~ 293

Author(s):

Yousuke Furuta ◽

Kazumi Takahashi ◽

Masako Kuno-Maekawa ◽

Hidehiro Sangawa ◽

Sayuri Uehara ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Mdck Cells ◽

Specific Inhibitor ◽

Polymerase Activity ◽

Virus Activity ◽

Virus Rna ◽

Cellular Dna

ABSTRACT T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5′-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5′-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.

Download Full-text

Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication

Journal of General Virology ◽

10.1099/vir.0.82199-0 ◽

2006 ◽

Vol 87 (11) ◽

pp. 3373-3377 ◽

Cited By ~ 29

Author(s):

Tao Deng ◽

Jane L. Sharps ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Replication Initiation ◽

Polymerase Complex ◽

Virus Rna ◽

Initiation Of Replication ◽

Rna Genome ◽

Transcription And Replication

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1–PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.

Download Full-text