Influenza A and B viruses have tremendous socioeconomic consequences, for influenza continues to occur in regular epidemics and occasional pandemics and is a leading cause of morbidity and mortality. The 2009 H1N1 pandemic was a wake-up call that influenza is still a continuing threat. Of continuing great concern today is the highly virulent avian H5N1 influenza virus that is still circulating among birds and transmitting to humans in parts of Far East Asia. Each year in the USA, in the absence of the introduction of pandemic influenza, seasonal influenza kills 36,000 people, hospitalizes 114,000 and causes 70 million missed work days and 38 million lost school days. It is estimated the loss to the economy is $3-15 billion. In the years of introduction of pandemic influenza virus, 1957 and 1967, approximately 70% of the US population was infected and in 1918/19 the estimate of death associated with Spanish influenza range from 20-40 million (1 in 100 people). Thus, there remains a need for efficient control of influenza virus by a new generation of vaccines or specific chemotherapeutics agents.
The aim of this grant application is to study the mechanism of influenza virus assembly. The direct relevance of this research to public health is that knowledge gained on the assembly mechanism of influenza virus provides new targets for anti-viral drug development. Among the least understood aspects of influenza virus replication are the events that allow the formation of virus particles and the pinching off o these particles by membrane fission. Recently, we have found a new role for the influenza virus M2 proton-selective ion channel protein in mediating virus budding. Our data indicate that an amphipathic helix (AH) in the M2 cytoplasmic tail mediates the final steps of budding for influenza viruses causing membrane scission and bypassing the need for using host cell proteins, as needed for budding of some other enveloped viruses e.g. HIV. We will perform a rigorous analysis of the function of the M2 cytoplasmic tail AH by generating a defined range of mutants in the AH and creating novel influenza viruses by reverse genetics. We will analyze the mutant viruses for changes in properties of the M2 protein function. We will analyze and reconstitute influenza virus budding in vitro and in vivo using assays that are well established in the field of membrane biophysics but are under utilized in the field of virology and that we have developed in our laboratory. We will analyze virus assembly in vivo by isolating membrane sheets. This permits, by using immuno-gold EM, examination of the temporal and spatial relationships between integral and membrane-associated viral proteins. This will provide major insight into the organization of the region of the plasma membrane involved in virus budding.

Public Health Relevance

Influenza virus remains an important viral pathogen of significant medical importance with pandemics occurring in 1918, 1957 and 2009: the 2009 H1N1 pandemic was a wake-up call that influenza is still a continuing threat and justifying that influenza virus is classified as a Select Agent Category C, because of its ability to cause great morbidity and mortality. Thus, there remains a need for efficient control of influenza virus by a new generation of vaccines or specific chemotherapeutics agents. The aim of this grant application is to study the mechanism of influenza virus assembly and the direct relevance of this research to public health is that knowledge gained on the assembly mechanism of influenza virus provides new targets for anti-viral drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020201-31
Application #
8605146
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hauguel, Teresa M
Project Start
1983-01-01
Project End
2017-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
31
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Evanston
State
IL
Country
United States
Zip Code
60201
Leser, George P; Lamb, Robert A (2017) Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane. J Virol 91:
Roberts, Kari L; Manicassamy, Balaji; Lamb, Robert A (2015) Influenza A virus uses intercellular connections to spread to neighboring cells. J Virol 89:1537-49
Wu, Yibing; Canturk, Belgin; Jo, Hyunil et al. (2014) Flipping in the pore: discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant S31N mutant of the influenza A virus M2 proton channel. J Am Chem Soc 136:17987-95
Rey-Carrizo, Matias; Barniol-Xicota, Marta; Ma, Chunlong et al. (2014) Easily accessible polycyclic amines that inhibit the wild-type and amantadine-resistant mutants of the M2 channel of influenza A virus. J Med Chem 57:5738-47
Bruns, Annie M; Leser, George P; Lamb, Robert A et al. (2014) The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly. Mol Cell 55:771-81
Rey-Carrizo, Matias; Torres, Eva; Ma, Chunlong et al. (2013) 3-Azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane derivatives: from wild-type inhibitors of the M2 ion channel of influenza A virus to derivatives with potent activity against the V27A mutant. J Med Chem 56:9265-74
Rossman, Jeremy S; Lamb, Robert A (2013) Viral membrane scission. Annu Rev Cell Dev Biol 29:551-69
Wang, Jizhou; Ma, Chunlong; Wang, Jun et al. (2013) Discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, S31N, of the M2 proton channel from influenza A virus. J Med Chem 56:2804-12
Roberts, Kari L; Leser, George P; Ma, Chunlong et al. (2013) The amphipathic helix of influenza A virus M2 protein is required for filamentous bud formation and scission of filamentous and spherical particles. J Virol 87:9973-82
Balgi, Aruna D; Wang, Jun; Cheng, Daphne Y H et al. (2013) Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay. PLoS One 8:e55271

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