Molecular Analysis of Alphavirus Membrane Fusion Protein

Project: Research project

Description

DESCRIPTION (provided by applicant): Alphaviruses and flaviviruses cause severe human and animal illnesses such as encephalitis, polyarthritis, and dengue fever, with millions of cases in humans per year. These viruses include many potential bioterrorist agents that are category A, B, or C priority pathogens, such as the encephalitic alphaviruses and the flaviviruses West Nile, yellow fever, Japanese encephalitis and dengue virus (DV). Alphaviruses and flaviviruses infect cells through a low pH-triggered membrane fusion reaction mediated by their structurally similar fusion proteins. These "class II fusion proteins" rearrange to a target-membrane inserted homotrimer (HT) to drive the fusion reaction. In collaboration with Dr. Felix Rey, we have recently determined the structure of the HT of the fusion protein E1 from the alphavirus Semliki Forest virus (SFV). This structure reveals that the HT is a trimeric hairpin in which domain III (DIII) and the stem region fold back against the trimer core, positioning the fusion peptide loops and transmembrane (TM) domains at the same side of the molecule. We have developed recombinant DIll proteins that block refolding to the final hairpin and inhibit SFV and DV fusion and infection. Based on this progress, we will now address the molecular mechanism of class II membrane fusion using the highly developed SFV experimental system. We will characterize the critical features of trimerization using expressed E1 domains and structure-based mutagenesis studies. DIll proteins and a wide variety of available virus fusion intermediates will be used to define key conformational changes and correlate them with steps in fusion. All of the post-fusion structures of viral fusion proteins are missing the TM domain, and thus its interaction with the fusion loops is undefined. Through our ongoing collaboration we will determine the structure of the full-length E1 HT. We will use cryo- and negative stain electron microscopy to define the interaction of the fusion loop with the target membrane and the specific contacts between HTs. We will test the role of HT-interactions both at the fusion site and outside the fusion site to address the functions of HT cooperativity and "bystander" fusion proteins. Understanding the molecular mechanism of class II membrane fusion will provide insights into virus disease mechanisms, enable the design of specific antiviral therapies, and advance our knowledge of viral and cellular membrane fusion reactions.
StatusActive
Effective start/end date8/15/9511/30/23

Funding

  • National Institutes of Health: $653,224.00
  • National Institutes of Health: $653,224.00
  • National Institutes of Health: $381,047.00
  • National Institutes of Health: $540,778.00
  • National Institutes of Health: $505,341.00
  • National Institutes of Health: $525,632.00
  • National Institutes of Health: $272,177.00
  • National Institutes of Health: $536,410.00
  • National Institutes of Health: $510,692.00
  • National Institutes of Health: $633,513.00
  • National Institutes of Health: $653,224.00
  • National Institutes of Health: $76,877.00
  • National Institutes of Health: $535,843.00
  • National Institutes of Health: $599,881.00
  • National Institutes of Health: $60,584.00

Fingerprint

Alphavirus
Membrane Fusion Proteins
Flavivirus
Semliki forest virus
Proteins
Membrane Fusion
Anethum graveolens
Nucleocapsid
Virus Internalization
Dengue
Viral Fusion Proteins
Viruses
Virus Diseases
Dissection
Membranes
Yellow Fever
Antiviral Agents
Japanese Encephalitis Virus
Rubella virus
West Nile Fever

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)