Viruses have many critical interactions with host cell membranes that mostly take advantage of host cell proteins. The Schirmer and Graham labs have 2 lines of investigation involving virus interactions with host cell membrane proteins that could be individually or jointly carried further in this PhD project.
The first regards nuclear egress, the least understood step in herpesviruses life cycles. Herpesviruses replicate and encapsidate their genomes in the host nucleus, making them too big to exit through nuclear pore complexes. They therefore bud through the inner nuclear membrane into the double-membrane nuclear envelope lumen, acquiring a primary envelope. These primary enveloped particles then fuse with the outer nuclear membrane to be released into the cytoplasm. We previously screened for host cell proteins that benefit this process, finding several vesicle fusion proteins of which we further characterised VAPB1. Knockdown of VAPB results in a ~2-log drop in virus infectivity and electron microscopy reveals accumulation of encapsidated virions in both the nucleus and lumen, suggesting functions in both envelopment and fusion steps. VAPB knockdown cells subjected to X-ray tomography revealed membrane blebs that suggested the exciting hypothesis that altered membrane curvature might prevent fusion with the primary enveloped particles. The student would develop skills in virus handling, identification of partner proteins, high-resolution microscopy, biophysics and image quantification in further investigating this hypothesis and also could test in more detail other host proteins identified in the screen.
The second project focuses on an observation that several nuclear membrane proteins accumulate in cytoplasmic puncta in SARS-CoV-2 infected cells. Previous pandemic coronaviruses SARS-CoV and MERS nsp3 and nsp4 proteins are core to the assembly of the double membrane vesicles (DMVs) thought to be where these viruses replicate their genomes2. We found expression of the SARS-CoV-2 nsp3 and nsp4 proteins could likewise generate DMVs and that these DMVs were defective when certain host cell nuclear envelope proteins were first depleted. This project would investigate the specific contributions of these host proteins to DMV formation, how they contribute to the efficiency of virus replication, and how their potentially structural function in the DMVs may parallel with their normal function in maintaining the double membrane structure of the nuclear envelope. This project will involve virus handling, comparing different coronaviruses and their nsp proteins, and different types of super resolution microscopy and protein interaction measurement tools in vivo such as FRET-FLIM.
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