Structure-Guided Creation of an Anti-HA Stalk Antibody F11 Derivative That Neutralizes Both F11-Sensitive and -Resistant Influenza A(H1N1)pdm09 Viruses

The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational rearrangements of HA for virus infection. Here we characterized molecular interactions between the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand the structural basis of the actions and modifications of this antibody. In silico structural analyses using a model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structure-guided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for the F11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivative that can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered conformational rearrangements and suggest that in silico guiding experiments can be useful to create anti-HA stalk antibodies with new phenotypes.

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Detailed Molecular Interactions of Favipiravir with SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza Virus Polymerases In Silico

Microorganisms ◽

10.3390/microorganisms8101610 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1610 ◽

Cited By ~ 1

Author(s):

Mitsuru Sada ◽

Takeshi Saraya ◽

Haruyuki Ishii ◽

Kaori Okayama ◽

Yuriko Hayashi ◽

...

Keyword(s):

Influenza Virus ◽

Molecular Interactions ◽

In Silico ◽

Influenza A ◽

Active Sites ◽

Active Form ◽

Antiviral Drug ◽

Virus Rna ◽

Influenza A H1n1 ◽

In Silico Studies

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5′-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.

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In Silico Identification of the Potential Drug Resistance Sites over 2009 Influenza A (H1N1) Virus Neuraminidase

Molecular Pharmaceutics ◽

10.1021/mp100041b ◽

2010 ◽

Vol 7 (3) ◽

pp. 894-904 ◽

Cited By ~ 51

Author(s):

Huanxiang Liu ◽

Xiaojun Yao ◽

Chengqi Wang ◽

Jian Han

Keyword(s):

Drug Resistance ◽

In Silico ◽

Influenza A ◽

H1n1 Virus ◽

Potential Drug ◽

Influenza A H1n1 ◽

In Silico Identification

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p‐cymene impairs SARS‐CoV‐2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS‐CoV‐2 nucleocapsid protein and H1N1 nucleoprotein

Pharmacology Research & Perspectives ◽

10.1002/prp2.798 ◽

2021 ◽

Vol 9 (4) ◽

Author(s):

Athanasios Panagiotopoulos ◽

Melpomeni Tseliou ◽

Ioannis Karakasiliotis ◽

Danai‐Maria Kotzampasi ◽

Vangelis Daskalakis ◽

...

Keyword(s):

Viral Replication ◽

In Silico ◽

Nucleocapsid Protein ◽

Influenza A ◽

Influenza A H1n1

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Structural basis and sequence co-evolution analysis of the hemagglutinin protein of pandemic influenza A/H1N1 (2009) virus

Experimental Biology and Medicine ◽

10.1258/ebm.2011.010264 ◽

2011 ◽

Vol 236 (8) ◽

pp. 915-925 ◽

Cited By ~ 8

Author(s):

Herman Tse ◽

Richard Y T Kao ◽

Wai Lan Wu ◽

Wilian W L Lim ◽

Honglin Chen ◽

...

Keyword(s):

Pandemic Influenza ◽

Influenza A ◽

Structural Basis ◽

Evolution Analysis ◽

Influenza A H1n1 ◽

Hemagglutinin Protein

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Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors

mBio ◽

10.1128/mbio.00430-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 16

Author(s):

Jeremy C. Jones ◽

Gyanendra Kumar ◽

Subrata Barman ◽

Isabel Najera ◽

Stephen W. White ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

Fold Change ◽

Influenza Viruses ◽

Single Amino Acid ◽

Structural Basis ◽

Next Generation ◽

Influenza A H1n1

ABSTRACT The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC 50 ]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC 50 ). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PA N target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro . Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs. IMPORTANCE The effectiveness of antiviral drugs can be severely compromised by the emergence of resistant viruses. Therefore, determination of the mechanisms by which viruses become resistant is critical for drug development and clinical use. RO-7 is a compound that potently inhibits influenza virus replication and belongs to a new class of drugs in late-stage clinical trials for treatment of influenza virus infection. Here we demonstrate that a single amino acid change acquired under prolonged virus exposure to RO-7 renders influenza viruses significantly less susceptible to its inhibitory effects. We have discovered how the mutation can simultaneously interfere with drug activity and still maintain efficient virus replication. These findings have important implications for the development of more effective derivatives of RO-7-like drugs and provide guidance for how to monitor the emergence of resistance.

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