Henipavirus assembly and budding

Open Access
Sun, Weina
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
February 25, 2015
Committee Members:
  • Anthony Paul Schmitt, Dissertation Advisor
  • Anthony Paul Schmitt, Committee Chair
  • Robert Paulson, Committee Member
  • Kumble Sandeep Prabhu, Committee Member
  • Pamela Hankey Giblin, Committee Member
  • Craig Eugene Cameron, Committee Member
  • Henipavirus
  • Hendra virus
  • Nipah virus
  • assembly and budding
  • paramyxovirus
  • AP-3 complex
  • Hendra virus fusion protein
  • Rab11a
The emergence of a group of paramyxoviruses called henipavirus has caused fatal illness such as severe vasculitis and encephalitis which resulted in high fatality rate in both humans and animals since 1990’s in south Asia and Australia. Like other paramyxoviruses, the essential viral protein that organizes the process of henipavirus assembly and budding is the matrix protein (M), where it functions to link together the viral glycoproteins and ribonucleoprotein complex (RNPs). Current studies have sought out to investigate the host factors that are involved in virus assembly and budding by interacting with M protein to facilitate M functions. In an effort to identify host factors that are important for henipavirus assembly and budding, proteomics-based approach was performed to identify host proteins that interact with viral M protein. Here, we affinity purified viral M proteins by FPLC and identified co-purifying host proteins by mass spectrometry. Co-immunoprecipitation was used as a secondary screening of protein candidates identified from mass spec results. One of the host proteins candidates, AP3B1, the beta subunit of AP-3 complex, was selected for further investigations. Importantly, a 29 amino acids polypeptide derived from AP3B1 hinge domain was identified as the minimal fragment to bind M protein as well as effectively inhibit henipavirus-like particles (VLPs) production. This inhibitory effect is due to disruption of M protein association with membrane. Additionally, in AP3B1 depleted cells by siRNA knockdown, Nipah VLP production is significantly reduced. By immunofluorescence iv microscopy, M protein colocalization with endogenous AP3B1 was also observed in mammalian cells. Our results suggested that AP-3 might play an important role in M protein functions. Henipaviruses have two types of surface glycoproteins, the attachment protein (G) and fusion protein (F). They were responsible for virus entry by mediating virus attachment to cell receptors and fusion with cell membrane. Numerous studies have illustrated how the virus entry occurs under the coordination of G and F proteins. However, little is known about how glycoproteins assemble into virions or VLPs. We know that M protein is the main driving force of particles formation, so we wondered how M protein coordinates with G protein and F protein to facilitate their trafficking and assembly into virions or VLPs. Here, under the unique pathway of HeV F protein identified by our collaborator Dr. Rebecca Dutch at University of Kentucky, we have found that HeV M protein, F protein and G protein all partially colocalized with Rab11a-REs, and overexpression of a DN Rab11a could significantly inhibit Hendra M-VLPs as well as F-VLPs formation in the cells. Interestingly, in cells that express inhibitory AP3B1 Hinge domain, M colocalization with Rab11-REs was disrupted. Unlike M protein or F protein, G proteins releases poorly into particles when expressed alone in cells. We observed that G incorporation into VLPs could be facilitated by co-expression of M protein but not F protein, likely through G-M interaction at the plasma membrane. Moreover, we have found that F protein incorporation into VLPs depended on its endocytic trafficking event regardless its cleavage status, as an endocytosis defective mutant F S490A that was retained on v the cell surface was shown to be significantly defective on particles assembly. On the other hand, HeV F protein still well incorporated into VLPs when its cleavage was prevented by a cathepsin L inhibitor, E-64d. Therefore, we hypothesized that henipavirus M and F proteins have separate mechanisms for trafficking to Rab11-REs, with the M protein trafficking facilitated by its interaction with AP3B1. M and cathepsin-cleaved F proteins must then assemble together within these compartments prior to their delivery to the cell surface for particle budding and assemble with G protein through G-M interaction at the plasma membrane.