We study the molecular mechanisms of human cytomegalovirus (HCMV) using cutting-edge mass spectrometry.
Human cytomegalovirus (HCMV) is a β-herpesvirus and a ubiquitous pathogen that infects over 60% of adults. HCMV infections are a significant cause of morbidity and mortality in immuno-compromised people, and can cause permanent hearing defects, vision loss, and mental retardation in newborn infants. Cells that are infected with HCMV undergo profound reorganization, and their cellular organelles are dramatically remodelled to sustain a structure known as the viral assembly complex (vAC). The vAC has been associated with packaging of the virus and extracellular release, however this process remains largely unknown.
As part of the ‘Infection and Immunity' theme in the Biomedicine Discovery Institute, research in our lab studies HCMV infection from both host and viral perspectives. We aim to identify host organelles that are targeted by viral proteins, and characterize the molecular mechanisms of HCMV using a hybrid multidiscipline approach integrating virology, molecular biology, microscopy, genomics, proteomics, and bioinformatics.
Research projects:
1) Dissecting the viral assembly complex
HCMV is a large double-stranded DNA virus whose 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. Importantly, there remains a gap in understanding how the virion is assembled, and the molecular mechanisms used by the virion to exit the infected cell (known as virial egress).
Infected cells undergo extensive organelle remodelling during viral infection that gives rise to the vAC. Several gross morphological alterations are induced including nuclear ‘kidney bean' morphology, and the Golgi and endosome components constitute a structure that resembles cylindrical rings of organelle-derived vesicles. This project investigates the following questions:
- How does the AC form?
- Which viral proteins are contained within the individual organelle vesicles?
- Which viral and host proteins are essential to the AC structure?
2) Revealing critical host-viral protein interactions mediating secondary envelopment
It is currently thought that to produce the mature virion, newly synthesized nucleocapsids containing viral DNA traffic through the nucleus and vAC, acquiring vrial tegument and glycoproteins. Prior to exit from infected cells, the outer virion envelope is thought to be derived by secondary membrane envelopment from the host, but the final cellular site where this occurs is still ambiguous. This project investigates the following questions:
- What is the organelle origin of the virion membrane?
- Which viral proteins are essential to secondary envelopment?
- What are the host-viral protein interactions that mediate this molecular mechanism?
3) Exploring the biological functions of the novel lipoamidase SIRT4
Sirtuins (SIRTs) are a critical family of seven mammalian nicotinamide adenine dinucleotide (NAD+)-dependent enzymes which govern genome regulation, metabolism, and aging. SIRTs display widespread subcellular distributions; SIRT1, SIRT6, and SIRT7 are nuclear, SIRT2 is predominantly cytoplasmic, and SIRTs3-5 are mitochondrial. Despite all SIRTs containing a conserved deacetylase domain, only SIRTs1-3 show robust deacetylation activity. Importantly, knock-down of SIRT expression, increases HCMV titer following infection, leading to their classification as viral restriction factors. However, the precise host mechanisms of action remain unknown.
We recently discovered SIRT4 is the first mammalian cellular lipoamidase, and can hydrolyse lipoyl- and biotinyl-lysine modifications far more efficiently than acetyl-lysine. Mitochondrial pyruvate dehydrogenase complex (PDH), which converts pyruvate to acetyl-CoA, is a biological substrate, and SIRT4 can enzymatically hydrolyze the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT) to diminish overall PDH activity. This project investigates the following questions:
- Can SIRT4 hydrolyze the lipoamide from other dehydrogenase complexes?
- Which biotin-modified mitochondrial proteins are substrates of SIRT4?
- Does HCMV have a protein that inhibits SIRT4 during infection?
Publications
* Denotes co-first author
22. Gopal S.K., Greening D.W., Hanssen E.G., Zhu H.J., Simpson R.J., and Mathias R.A. (2016) Oncogenic epithelial cell-derived exosomes containing Rac1 and PAK2 induce angiogenesis in recipient endothelial cells. Oncotarget.
21. Mathias R.A., Greco T.M., and Cristea I.M. (2016) Identification of SIRT4 protein interactions: Uncovering candidate acyl-modified mitochondrial substrates and enzymatic regulators. Methods Mol. Biol.
20. Gopal S.K., Greening D.W., Mathias R.A., Ji H., Rai A., Chen M., Zhu H.J., and Simpson R.J. (2015) YBX1/YB-1 induces partial EMT and tumourigenicity through secretion of angiogenic factors into the extracellular microenvironment. Oncotarget 6:13718-13730
19. Mathias R.A., Guise A.J., and Cristea I.M. (2015) Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease. Mol. Cell. Proteomics 14:456-470
18. Greening D.W., Gopal S.K., Mathias R.A., Liu L., Sheng J., Zhu H.J., and Simpson, R.J. (2015) Emerging roles of exosomes during epithelial-mesenchymal transition and cancer progression. Semin. Cell Dev. Biol. 40:60-71
17. Mathias R.A., Greco T.M., Oberstein A., Budayeva H.G., Chakrabarti R., Rowland E.A., Kang Y., Shenk T., and Cristea I.M. (2014) Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity. Cell 159:1615-1625
16. Guise A.J., Mathias R.A., Rowland E.A., Yu F., and Cristea I.M. (2014) Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5). Proteomics 14:2156-2166
*15. Tauro B.J., Mathias R.A., Greening D.W., Gopal S.K., Ji H., Kapp E.A., Coleman B.M., Hill A.F., Kusebauch U., Hallows J.L., Shteynberg D., Moritz R.L., Zhu H.J., and Simpson R.J. (2013) Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition. Mol. Cell. Proteomics 12:2148-2159
14. Tauro B.J., Greening D.W., Mathias R.A., Mathivanan S., Ji H., and Simpson R.J. (2013) Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Mol. Cell. Proteomics 12:587-598
13. Mathias R.A., Gopal S.K., and Simpson R.J. (2013) Contribution of cells undergoing epithelial-mesenchymal transition to the tumour microenvironment. Journal of proteomics 78:545-557
12. Tauro B.J., Greening D.W., Mathias R.A., Ji H., Mathivanan S., Scott A.M., and Simpson R.J. (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293-304
11. Mathias R.A., Ji H., and Simpson R.J. (2012) Proteomic profiling of the epithelial-mesenchymal transition using 2D DIGE. Methods Mol. Biol. 854:269-286
10. Lim J.W., Mathias R.A., Kapp E.A., Layton M.J., Faux M.C., Burgess A.W., Ji H., and Simpson R.J. (2012) Restoration of full-length APC protein in SW480 colon cancer cells induces exosome-mediated secretion of DKK-4. Electrophoresis 33:1873-1880
9. Mathias R.A., Chen Y.S., Kapp E.A., Greening D.W., Mathivanan S., and Simpson R.J. (2011) Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins. Methods 54:396-406
8. Mathias R.A., Chen Y.S., Goode R.J., Kapp E.A., Mathivanan S., Moritz R.L., Zhu H.J., and Simpson R.J. (2011) Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database. Proteomics 11:1238-1253
7. Ji H., Goode R.J., Vaillant F., Mathivanan S., Kapp E.A., Mathias R.A., Lindeman G.J., Visvader J.E., and Simpson R.J. (2011) Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations. Proteomics 11:4029-4039
*6. Chen Y.S., Mathias R.A., Mathivanan S., Kapp E.A., Moritz R.L., Zhu H.J., and Simpson R.J. (2011) Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition. Mol. Cell. Proteomics 10:M110 001131
5. Bernhard O.K., Mathias R.A., Barnes T.W., and Simpson R.J. (2011) A fluorescent microsphere-based method for assay of multiple analytes in plasma. Methods Mol. Biol. 728:195-206
4. Mathias R.A., Chen Y.S., Wang B., Ji H., Kapp E.A., Moritz R.L., Zhu H.J., and Simpson R.J. (2010) Extracellular remodelling during oncogenic Ras-induced epithelial-mesenchymal transition facilitates MDCK cell migration. J. Proteome Res. 9:1007-1019
3. Mathias R.A., Wang B., Ji H., Kapp E.A., Moritz R.L., Zhu H.J., and Simpson R.J. (2009) Secretome-based proteomic profiling of Ras-transformed MDCK cells reveals extracellular modulators of epithelial-mesenchymal transition. J. Proteome Res. 8:2827-2837
2. Mathias R.A., and Simpson R.J. (2009) Towards understanding epithelial-mesenchymal transition: a proteomics perspective. Biochim. Biophys. Acta 1794:1325-1331
1. Mathias R.A., Lim J.W., Ji H., and Simpson R.J. (2009) Isolation of extracellular membranous vesicles for proteomic analysis. Methods Mol. Biol. 528:227-242
