Elucidating the role of host factors in henipavirus budding
Open Access
- Author:
- Sabahat Gazal, Fnu
- Graduate Program:
- Pathobiology (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 23, 2021
- Committee Members:
- Anthony Schmitt, Chair & Dissertation Advisor
Robert Paulson, Major Field Member
Troy Sutton, Major Field Member
Joyce Jose, Outside Unit & Field Member
Anthony Schmitt, Program Head/Chair - Keywords:
- Henipavirus
ESCRT
angiomotins
Nedd4L
VLPs - Abstract:
- Nipah and Hendra viruses are highly virulent, zoonotic BSL-4 agents that emerged in 1990’s and since then have been responsible for causing hundreds of human fatalities throughout Asia and Australia. Henipaviruses belong to family Paramyxoviridae and have been found to depend on ESCRT machinery to facilitate live virus budding. However, previous studies have shown that henipavirus M VLP budding is ESCRT independent. In this study, we showed that henipavirus F VLP budding is ESCRT dependent and ubiquitin dependent. We further showed the Nedd4 family ubiquitin ligase, Nedd4L enhances henipavirus F VLP budding and that ablation of ubiquitin ligase activity of Nedd4L inhibits F VLP budding. These findings provide first indication that in case of henipaviruses, Nedd4L is recruited by a viral transmembrane glycoprotein to facilitate budding in an ESCRT dependent manner. For some paramyxoviruses like PIV5 and mumps virus (that lack any of the conventional late domains), a host protein named angiomotin like 1 (AmotL1) has been found to play an important role in budding by acting as a bridge between the viral M protein and Nedd4 family ubiquitin ligases. Since henipaviruses also lack any of the well characterized late domains and as we found henipavirus F VLP budding to be ESCRT and ubiquitin dependent, the study further aimed to determine the role of angiomotins in henipavirus F VLP budding. Angiomotin family of proteins include angiomotin (Amot) and angiomotin like proteins 1 and 2 viz. AmotL1 and AmotL2. Using CRISPR/ Cas9 mediated knockout of Amot or AmotL1, we showed that knockout of angiomotins inhibit henipavirus F VLP budding but had no significant effect on M VLP budding. Double knockout of Amot and AmotL1 resulted in near complete inhibition of F VLP budding. Angiomotins have PPXY domains through which they interact with Nedd4 family ubiquitin ligases. Here we have shown that the PPXY domains of AmotL1 play an important role in Hendra virus F VLP budding as the overexpression of mutant AmotL1 lacking LPTY and PPXY sequences lead to inhibition of Hendra virus F VLP budding. Co-localization studies showed that GFP fused to cytoplasmic tail of Hendra virus F co-localizes with AmotL1 and also Nedd4L. We propose that henipavirus F proteins instead of directly harboring PPXY sequences, bind to PPXY containing angiomotins and thereby recruit Nedd4L and thus ESCRT in a way similar to that observed with viruses harboring a PPXY late domain.