Protein P7 of phage φ6 RNA polymerase complex, acquiring of RNA packaging activity by In Vitro assembly of the purified protein onto deficient particles

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

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Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the aroP Gene ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.180.20.5466-5472.1998 ◽

1998 ◽

Vol 180 (20) ◽

pp. 5466-5472 ◽

Cited By ~ 17

Author(s):

Peixiang Wang ◽

Ji Yang ◽

Akira Ishihama ◽

A. J. Pittard

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Regulatory Region ◽

Ofescherichia Coli ◽

Mrna Synthesis ◽

Polymerase Complex ◽

Defective Mutant ◽

Dna Fragment ◽

Region Ii

ABSTRACT In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

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The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes

Nucleic Acids Research ◽

10.1093/nar/gkm1044 ◽

2007 ◽

Vol 36 (1) ◽

pp. 245-252 ◽

Cited By ~ 14

Author(s):

Sven Nottebaum ◽

Lin Tan ◽

Dominika Trzaska ◽

Hannah C. Carney ◽

Robert O. J. Weinzierl

Keyword(s):

Recombinant Protein ◽

Rna Polymerase ◽

High Throughput ◽

Protein Complexes ◽

In Vitro Assembly

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Influenza A virus proteins PB1 and NS1 are subject to functionally important phosphorylation by protein kinase C

Journal of General Virology ◽

10.1099/vir.0.009050-0 ◽

2009 ◽

Vol 90 (6) ◽

pp. 1392-1397 ◽

Cited By ~ 19

Author(s):

Shohreh Mahmoudian ◽

Sabrina Auerochs ◽

Monika Gröne ◽

Manfred Marschall

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Rna Polymerase ◽

Influenza A ◽

Polymerase Activity ◽

Viral Rna ◽

Influenza A Viruses ◽

Polymerase Complex ◽

Kinase C

The virulence of influenza A viruses depends on the activity of the viral RNA polymerase complex and viral regulatory phosphoproteins. We identified that the protein kinase C (PKC) inhibitor Gö6976 had a post-entry anti-influenza viral effect, by using a polymerase activity-based reporter assay. This inhibitory effect was observed for influenza virus-infected cells as well as for cells transiently transfected with constructs for the RNA polymerase complex. Importantly, the in vitro analysis of viral protein phosphorylation identified PKCα as a kinase phosphorylating PB1 and NS1, but not PB2, PA or NP. Gö6976 was able to block PKC-specific phosphorylation in vitro. Thus, our data suggest that PKC contributes to the phosphorylation of influenza PB1 and NS1 proteins which appears to be functionally relevant for both viral RNA polymerase activity and efficient viral replication.

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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.

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Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase complex activity: Synthesis and in vitro evaluation

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2007.06.084 ◽

2007 ◽

Vol 17 (18) ◽

pp. 5199-5203 ◽

Cited By ~ 38

Author(s):

Aiming Sun ◽

Nizal Chandrakumar ◽

Jeong-Joong Yoon ◽

Richard K. Plemper ◽

James P. Snyder

Keyword(s):

Rna Polymerase ◽

Measles Virus ◽

Complex Activity ◽

Rna Dependent Rna Polymerase ◽

In Vitro Evaluation ◽

Polymerase Complex ◽

Virus Rna ◽

Nucleoside Inhibitors

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Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6

Journal of Virology ◽

10.1128/jvi.72.12.10058-10065.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10058-10065 ◽

Cited By ~ 21

Author(s):

Anja O. Paatero ◽

Leonard Mindich ◽

Dennis H. Bamford

Keyword(s):

Rna Polymerase ◽

Mutational Analysis ◽

Rna Virus ◽

Nucleoside Triphosphate ◽

Particle Assembly ◽

Polymerase Complex ◽

Translocation Event ◽

Shell Forming ◽

Nucleoside Triphosphatase

ABSTRACT Bacteriophage φ6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.

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Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

10.1101/2020.12.14.422718 ◽

2020 ◽

Author(s):

Jack PK Bravo ◽

Tyler L Dangerfield ◽

David W Taylor ◽

Kenneth A Johnson

Keyword(s):

Rna Polymerase ◽

Active Site ◽

Rational Design ◽

Antiviral Drugs ◽

Chain Termination ◽

Nucleoside Analog ◽

Rna Dependent Rna Polymerase ◽

Polymerase Complex

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

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