An effector of amphibian innate immunity protects against human influenza A viruses 公开
Holthausen, David (Fall 2017)
Published
Abstract
While the current program of influenza vaccines is effective at conferring protection against influenza A viruses, varying from 19-60%, antiviral treatments are a necessary second line of defense when vaccine mismatch and pandemic outbreaks occur. With the increased reliance on the current antiviral drugs, there is also an increased susceptibility to the emergence of drug resistant viral strains. As such, it is crucial to develop new antiviral therapies. We studied a host defense peptide secreted from the skin of the South Indian frog Hydrophylax bahuvistara. This peptide, which we named “urumin”, is virucidal for H1 hemagglutinin-bearing influenza viruses. Urumin specifically targets the conserved stalk domain of H1 hemagglutinin and demonstrates effectiveness against drug-resistant H1 influenza viruses. Electron microscopy studies demonstrated that urumin physically destroys influenza virions. Urumin also protected against lethal influenza infection in vivo.
To determine the mechanism of urumin’s antiviral activity, we conducted bioinformatic analysis and 3-dimensional modeling of hemagglutinin. We identified an extremely conserved amino acid loci, 417L, in the stalk of HA that is crucial for urumin activity. Using an A/Memphis/8/2003 H1N2 influenza virus, which contains an L417F mutation, we found that urumin’s antiviral activity is ablated. When this L417F mutation is inserted into a urumin-susceptible virus, A/Puerto Rico/8/1934, antiviral activity is absent. Western blot analyses demonstrated that urumin acts by separating the HA trimer and disrupting the disulfide linkage between HA1 and HA2. This activity is dependent upon urumin having free cysteine residues. Given the mechanism of action, and the nature of the HA cleavage site, we speculate that peptide modification strategies can improve urumin activity against influenza subtypes other than H1.
Urumin represents a unique class of anti-influenza virucide, acting like a thiol reductase, specifically targeting the HA disulfide bridge, bonding to HA1, and releasing HA2. These findings indicate the potential for urumin as a clinical anti-influenza therapeutic, especially given that peptide modification strategies should enhance effectiveness against other influenza strains. Overall, these studies establish a novel mechanism of anti-influenza activity, and demonstrate the importance of research into non- human host defense peptides.
Table of Contents
ABSTRACT ............................................................................................................... IV
ACKNOWLEDGEMENTS .......................................................................................VII
CHAPTER 1: INTRODUCTION ................................................................................ 1
GLOBAL AND FINANCIAL IMPACT OF INFLUENZA.........................................................................1
Figure 1: Map of Worldwide Influenza Transmission ................................................................2
INFLUENZA A VIRAL STRUCTURE AND FUNCTION........................................................................3
Figure 2: Influenza A Virus Structure ........................................................................................ 4
INFLUENZA INFECTION, PATHOGENESIS, AND VIRULENCE ..........................................................9
INFLUENZA VACCINES, ANTIVIRALS, AND RESISTANCE ..............................................................13
HOST DEFENSE PEPTIDES.............................................................................................................16
Figure 3: Shai-Matsuzaki-Huang Model of Host Defense Peptide Mechanism.........................20
AMPHIBIAN HOST DEFENSE PEPTIDES.........................................................................................23
ANTI-INFLUENZA PEPTIDES ...........................................................................................................25
SUMMARY .....................................................................................................................................29
REFERENCES .................................................................................................................................30
CHAPTER 2: AN AMPHIBIAN HOST DEFENSE PEPTIDE IS VIRUCIDAL FOR HUMAN H1 HEMAGGLUTININ-BEARING INFLUENZA VIRUSES ... 53
AUTHORS ......................................................................................................................................53
AFFILIATIONS................................................................................................................................53
SUMMARY .....................................................................................................................................54
INTRODUCTION ............................................................................................................................54
RESULTS ........................................................................................................................................56
Figure 1. A Peptide from Hydrophylax bahuvistara Exhibits Anti-A/PR/8/1934 Influenza Virus Activity In Vitro.......58
Figure 2. Urumin Is Specific for H1 Hemagglutinin, Targets the Conserved Stalk Region of H1 HA, and Is Effective against Drug-Resistant Influenza Viruses.......62
Figure 3. Urumin Disrupts Influenza Virus Integrity and Requires Sequence Fidelity and Chirality for Activity.......65
Figure 4. Intranasal Administration of urumin Reduces Influenza-Induced Morbidity, Mortality, and Lung Viral Titers In Vivo.................................................................................68
DISCUSSION ....................................................................69
EXPERIMENTAL PROCEDURES ......................................................................................................71
SUPPLEMENTAL INFORMATION ....................................................................................................75
Figure S1. Peptides that demonstrate anti-A/PR/8/1934 activity in vitro .............................75
Figure S2. Increasing urumin peptide concentration leads to increased virus growth inhibition. ........76
Figure S3. Alanine scan mutants and D enantiomer of urumin peptide are not toxic to human red blood cells..............77
AUTHOR CONTRIBUTIONS ...........................................................................................................78
ACKNOWLEDGEMENTS ................................................................................................................78
REFERENCES .................................................................................................................................79
CHAPTER 3: THE VIRUCIDAL PEPTIDE URUMIN ACTS LIKE A THIOL REDUCTASE, BINDS TO AND DESTABILIZES HEMAGGLUTININ ON HUMAN INFLUENZA VIRUSES ............................................................................................. 84
AUTHORS ......................................................................................................................................84
AFFILIATIONS................................................................................................................................84
SUMMARY .....................................................................................................................................85
INTRODUCTION ............................................................................................................................85
RESULTS ........................................................................................................................................87
Figure 1. Position 417L on the stalk region of H1 hemagglutinin is an extremely conserved and accessible locus at the center of HA ..........88
Figure 2. Urumin does not inhibit the H1N2 A/Memphis/8/2003 virus containing an L417F mutation and is effective against PR8 mutant viruses that retain a leucine at position 417 on the HA stalk ..........................92
Figure 3. Urumin acts as a reducing agent to physically disrupt hemagglutinin........................96
Figure 4. Urumin acts like a thiol reductase, cleaves the HA disulfide bond, and remains bound to the HA1 subunit ...........98
DISCUSSION ..................................................................................................................................99
EXPERIMENTAL PROCEDURES ................................................................................................... 101
SUPPLEMENTAL INFORMATION ................................................................................................. 104
Figure S1. Sequence analysis of A/Memphis/8/2003 H1N2 HA against 8 HA from H1N1 influenza viruses. Related to Figure 2. .........104
Figure S2. Urumin is effective but has reduced activity at low temperatures. Related to Figure 3. ........105
AUTHOR CONTRIBUTIONS ........................................................................................................ 105
ACKNOWLEDGEMENTS ............................................................................................................. 106
REFERENCES .............................................................................................................................. 106
CHAPTER 4: DISCUSSION.................................................................................... 110
URUMIN AS AN INFLUENZA ANTIVIRAL THERAPY .................................................................... 110
MODELING A NOVEL MECHANISM OF ANTI-INFLUENZA ACTIVITY ........................................ 115
THE UNTAPPED RESOURCE OF HOST DEFENSE PEPTIDES ....................................................... 119
FUTURE DIRECTIONS................................................................................................................. 120
REFERENCES .............................................................................................................................. 122
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