Hendra virus (HeV) and Nipah virus, members of the Henipavirus genus of the paramyxovirus family, are zoonotic viruses with high mortality rates in humans. Henipavirus particle assembly is coordinated by the viral matrix (M) proteins, which link together the viral glycoproteins and the viral ribonucleoproteins. The Dutch lab recently defined a novel mechanism for proteolytic cleavage of the Henipavirus fusion (F) proteins. In contrast to other paramyxovirus F proteins, Henipavirus F proteins reach the cell surface in the inactive, uncleaved form, are endocytosed from the plasma membrane (PM), and encounter cathepsin proteases within early endosomes (EEs). Following cathepsin cleavage, F is routed through Rab11-containing recycling endosomes (REs) before returning to the PM for incorporation into budding virus particles. The Schmitt lab recently identified interactions between Henipavirus matrix (M) proteins and the beta subunit of AP-3 adapter complexes which are involved in endosomal trafficking. M protein localized in Rab11 compartments, and this localization was disrupted upon expression of M- binding AP3B1-derived inhibitory polypeptides. To explore the significance of M and F protein convergence in Rab11-positive REs, the Dutch and Schmitt labs have now combined their efforts and discovered that an F mutant defective in endocytic trafficking fails to participate in VLP assembly, and that introduction of a dominant negative Rab11 protein dramatically inhibits Hendra VLP production. We therefore hypothesize that Rab-11 containing endosomal compartments serve as a critical assembly site for key viral proteins prior to viral budding.
In Aim 1, we will examine the role of HeV virus F protein trafficking in vius assembly, through use of a comprehensive set of trafficking-altered F protein mutants, and will analyze F- M interactions in Rab11 REs using a sensitive proximity ligation assay.
Our second aim will build on the exciting preliminary data defining an interaction between HeV M protein and the beta subunit of the AP-3 adapter complex, examining the role of this interaction in M trafficking, delineating the trafficking pathway, and examining the role of this interaction in apial targeting of the HeV M protein. Finally, in Aim 3 we will address the role of HeV G protein interactions with M or F on G assembly, testing the hypothesis that G protein assembly into VLPs is mediated by redundant interactions with M and F proteins. These experiments will provide critical new information on HeV assembly, and test the novel hypothesis that Rab11 REs may serve as viral assembly sites. If this hypothesis is correct, Rab11 RE components involved in membrane bending may serve as novel host factors for non-ESCRT mediated budding.

Public Health Relevance

The highly pathogenic Hendra and Nipah viruses are recently emerged human pathogens for which no vaccines or antivirals for human use are available. The proposed experiments will provide new understanding of the process of Hendra virus assembly and budding, and could lead to development of new antiviral reagents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI108260-01A1
Application #
8722208
Study Section
Special Emphasis Panel (ZRG1-IDM-S (02))
Program Officer
Cassetti, Cristina
Project Start
2014-02-10
Project End
2016-01-31
Budget Start
2014-02-10
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$211,873
Indirect Cost
$45,099
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
El Najjar, Farah; Schmitt, Anthony P; Dutch, Rebecca Ellis (2014) Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production. Viruses 6:3019-54