HCMV is a herpesvirus that infects over 60% of the adult population. HCMV is a significant cause of morbidity and mortality in immuno-compromised individuals, such as organ transplant recipients. Additionally, the largest burden of disease occurs from intrauterine transmission during pregnancy. This occurs in greater than 1% of pregnancies worldwide, and can cause permanent hearing loss, vision impairment, and mental retardation. There is no vaccine currently available, and discovery of new antivirals is urgently required. Importantly, the process by which infectious virus is packaged and released is not well understood, and this presents a novel molecular axis to develop antiviral therapeutics. RESEARCH STRATEGY Research in our laboratory uses a multidisciplinary approach to better understand host defense mechanisms, and identify cellular pathways that are hijacked by HCMV. We work at the interface between cell biology, virology and quantitative proteomics, and have proprietary virus libraries and reagents to make unique discoveries. All projects have an opportunity to learn standard (tissue culture, western blotting, immune-precipitation, confocal microscopy, RNAi, CRISPR) and advanced (liquid chromatography, mass spectrometry, bioinformatics, electron microscopy) laboratory techniques and skills. PROJECT 1: Biogenesis of the HCMV viral assembly complex HCMV is a large double-stranded DNA virus whose 236 kbp genome is known to code for at least 150 proteins. The HCMV virion comprises a nucleocapsid that houses the DNA genome, and is surrounded by a proteinaceous tegument layer, and glycoprotein-containing lipid envelope. During infection, exactly how the virion is assembled and released (egress) remains unknown. However, infection causes extensive organelle remodeling in infected cells, and produces a structure known as the viral assembly complex (vAC). It is currently thought that the vAC facilitates virion assembly and maturation. We have a library of mutant viruses that contain a transposon to disrupt each of the 150 open reading frames in the HCMV genome. We use these mutant viruses to identify the essential viral proteins needed to generate the vAC. Using a confocal microscopy-based assay, we screen for defects in Golgi ring, endosome clustering, and secondary envelopment of the virion. Ongoing efforts in the lab have revealed 9 candidate viral proteins that regulate secondary envelopment. Many of these are novel viral proteins with uncharacterized functions, and await further investigation. PROJECT 2: Hijacking of host exosome pathways by HCMV HCMV is master at manipulating existing host pathways to benefit completion of the life cycle. It is known that maturing nucleocapsids bud into host membrane-derived structures to acquire the outer virion envelope. However, the origin of the membrane is unknown, and the precise molecular mechanisms remain elusive. In our lab, proteomic sequencing identified a strong enrichment of host exosome proteins in the virion. Exosomes are small nanovesicles (50-200 nm) secreted by almost all cell types. Therefore, we hypothesize that HCMV hijacks this pathway for viral egress. We use CRISPR to knock-out various host exosome proteins in cells, and measure the functional impact to virions by assaying viral titre released from these ‘edited’ cells. Incorporation of host exosome proteins into infectious virions is also validated by immuno- gold EM. Thus far, our investigations have unearthed the tetraspanins as a family of host proteins that facilitate virion assembly, and will be explored further.
Virus, Host, Virology, Infection, Proteomics, Cell biology, Molecular, Interaction, Microbiology, Biochemistry, Mass spectrometry, Trafficking, Secretion, Department of Microbiology
Biomedicine Discovery Institute (School of Biomedical Sciences) » Microbiology
Masters by research
Top-up scholarship funding available
18 Innovation Walk