Infection by the human paramyxovirus parainfluenza virus (HPIV) causes major respiratory diseases, including croup, bronchiolitis and pneumonia, which lead to illness or death in millions of infants and young children worldwide. HPIV infects by fusing its envelope directly with the host cell membrane. During entry, the viral surface glycoproteins HN (receptor-binding protein;hemagglutinin-neuraminidase) and F (fusion protein) cooperate to mediate fusion upon receptor binding. However, until an incoming virus meets its target cell, the fusion machine is inactive. Each step in viral entry intimately reflects the natural host cell surface;to understand -- and to interfere with -- natural infection, we consider the fundamental mechanisms that affect virus-cell interplay in the host. The long-term goal of our laboratory is to understand HPIV entry at a level of detail that permits design of antiviral drugs that hijack the HN-F machinery, and to understand the mechanistic commonalities among the fusion machineries of paramyxoviruses. The Objective of This Application is to define the mechanisms of the paramyxovirus HN-F machinery that are relevant for infection in the human.
Aim 1. Determine HN's role in preventing premature activation of F-mediated fusion. The HPIV3 HN-F fusion machinery must be activated only at the right place and time. We will determine whether HN protects F from being triggered prematurely, and whether this role is critical in the natural host, preventing production of inactive viral particle and abortive infection. Using full-length mutant HN proteins and recombinant chimeric HN proteins, we will identify the domain(s) of HN that retain F in its pre-triggered state, and define the mechanisms of HN's protective role.
Aim 2. Identify the key interactions between native HN and F that result in timely activation of F. Using infectious virus and engineered liposomes, we will capture serial intermediate states of the HPIV3 fusion complex during activation, to identify the structural rearrangements in the HN-F complex in its native state during the fusion process. Using mutant and chimeric HN-F complexes, we will define the determinants for HN's activation of F, and for HN's role in shepherding F through a series of transient intermediates in an ongoing HN-F complex, in native viruses and in real time.
Aim 3. The HN-F fusion machine is tailored to the natural host: Identify the essential features of this virus-host interplay during vral entry, and exploit them for design of novel antiviral targets. Circulating HPIV3 viruses require a balance of HN-F fusion machinery properties that results in an inverse correlation between fusion in cultured cells and growth in vivo. This requirement can be hijacked for an effective antiviral strategy. Human clinical HPIV3 isolates will be used to determine both the required features of the HN-F fusion machinery that determine viability in vivo and whether perturbing the HN-F fusion machinery affects infection in vivo. Overall, our studies will move the picture of paramyxovirus infection to a paradigm relevant for human infection, providing the scientific basis for the first treatments for HPIV.

Public Health Relevance

Acute respiratory infection is now the leading cause of mortality in young children under 5 years of age, accounting for nearly one fifth (20%) of childhood deaths worldwide, and killing 2-3 million children each year. Human parainfluenza viruses are a major cause of childhood croup, bronchiolitis and pneumonia in the U.S. Despite the huge impact of these diseases on illness and hospitalization of young children worldwide, no drugs or vaccines are available. This research proposal will lead to a better understanding of how paramyxoviruses enter human cells to initiate infection. Newly identified mechanisms involved in the entry process, now considered in the proper biological context to include clinically circulating strains and human airway models, could serve as new targets for antivirals to treat or prevent human respiratory diseases. Effective therapy for pediatric respiratory viruses would significantly decrease morbidity and mortality due to lower respiratory disease for children, the elderly and the immunocompromised in the U.S. and worldwide.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI031971-22
Application #
8662676
Study Section
Virology - B Study Section (VIRB)
Program Officer
Kim, Sonnie
Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065
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Talekar, Aparna; DeVito, Ilaria; Salah, Zuhair et al. (2013) Identification of a region in the stalk domain of the nipah virus receptor binding protein that is critical for fusion activation. J Virol 87:10980-96
Xu, Rui; Palmer, Samantha G; Porotto, Matteo et al. (2013) Interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type III regulates viral growth in vivo. MBio 4:e00803-13
Talekar, Aparna; Moscona, Anne; Porotto, Matteo (2013) Measles virus fusion machinery activated by sialic acid binding globular domain. J Virol 87:13619-27
Porotto, Matteo; Salah, Zuhair; DeVito, Ilaria et al. (2012) The second receptor binding site of the globular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion. J Virol 86:5730-41
Porotto, Matteo; Palmer, Samantha G; Palermo, Laura M et al. (2012) Mechanism of fusion triggering by human parainfluenza virus type III: communication between viral glycoproteins during entry. J Biol Chem 287:778-93
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Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G et al. (2011) Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein. J Virol 85:12867-80
Moscona, Anne (2007) CSI microbiology: emerging pathogens and a staged strategy for detection and discovery. J Infect Dis 196:1727-8

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