PhD candidate in molecular life science at Monash University
Invisible, inaudible; we hold the enemy's fate in our hands
Creatively channelling the strengths of an apparently insurmountable foe has long been said to be the key to defeating it.
Since being first isolated in 1983, the human immunodeficiency virus (HIV) has spread rapidly and is now carried by approximately 0.5% of the world’s population. Furthermore, HIV can replicate within a patient as many as 100-times the world’s population in new virus particles in a single day, leading to large genetic changes within the virus and enabling resistant strains to emerge. However, 100% of these particles require human proteins to assist the virus to move its components throughout the host cell in order to replicate. These proteins form part of the viral ‘command and control’ mechanism, enabling host cell processes to be redirected in a manner favourable to the virus.
How might we use the unique abilities of viruses such as HIV to our own therapeutic advantage?
My research focuses on understanding how these human proteins move between different areas of the cell and their exquisite functions therein. One of these ways is through a barcoding process known as ‘post-translational modification’, where proteins are tagged with one of hundreds of different molecules that alter their function, abundance or distribution in the cell. My work combines a suite of technologies to perform detailed analysis of specific DNA, RNA or protein sequences with large-scale multivariate analyses to track changes in the interplay between host and virus in space and time. Equipped with this knowledge, I seek to understand why and how viruses manipulate these proteins to their own advantage, with the aim of dismantling their mechanisms of cellular command and control through minimally invasive means.
My vision is to leverage one of the greatest evolutionary strengths of pandemic viruses, namely their ability to rapidly mutate and co-evolve with their host, to create and begin a new way of designing antiviral therapies that are ultimately cost-effective yet resilient against virus escape mutants. If this vision resonates with you also, then please take a moment to follow my pinboard and join in this exciting journey.
Abstract: Viral infection activates danger signals that are transmitted via the retinoic acid-inducible gene 1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and Toll-like receptor (TLR) protein signaling cascades. This places host cells in an antiviral posture by up-regulating antiviral cytokines including type-I interferon (IFN-I). Ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate IFN-I expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress IFN-I. Here we review how host- and virus-directed ubiquitin modification of proteins in the RLR, NLR, and TLR antiviral signaling cascades modulate IFN-I expression.
Pub.: 30 Dec '15, Pinned: 28 Aug '17
Abstract: Interferon-stimulated gene (ISG) products take on a number of diverse roles. Collectively, they are highly effective at resisting and controlling pathogens. In this review, we begin by introducing interferon (IFN) and the JAK-STAT signaling pathway to highlight features that impact ISG production. Next, we describe ways in which ISGs both enhance innate pathogen-sensing capabilities and negatively regulate signaling through the JAK-STAT pathway. Several ISGs that directly inhibit virus infection are described with an emphasis on those that impact early and late stages of the virus life cycle. Finally, we describe ongoing efforts to identify and characterize antiviral ISGs, and we provide a forward-looking perspective on the ISG landscape.
Pub.: 22 Feb '14, Pinned: 28 Aug '17
Abstract: BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species.
Pub.: 11 Feb '14, Pinned: 28 Aug '17
Abstract: This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome-wide (microarrays) and targeted (real-time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target-specific, color-coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35- to 50-base target-specific sequences. In addition, each reporter probe carries a unique color code at the 5' end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3' end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution-phase hybridization between target mRNA and reporter-capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color-coded barcode for that gene is detected, and the barcode counts are then tabulated.
Pub.: 08 Apr '11, Pinned: 28 Aug '17
Abstract: Human immunodeficiency virus (HIV) spread to humans from chimpanzees (HIV-1 groups M and N), gorillas (HIV-1 groups P and O), and sooty mangabeys (HIV-2). HIV is spread mainly through blood or body fluids. Subjects can become infected with HIV by sexual contact, needle sharing, blood transfusions, or maternal transmissions as a blood-borne virus or via breast-milk. The incubation period of HIV-1 from infection to the development of AIDS ranges from 8 to 11 years. In the past 3 decades, HIV has caused a great burden to global wealth and health. According to the WHO global health survey, 36.7 million people were infected with HIV, causing 1.1 million deaths in 2015. Since the discovery of HIV-1, many anti-retroviral drugs have been developed. Following the discovery and wide-spread use of anti-retroviral therapy (ART) the life expectancy of HIV infected individuals has substantially increased. By 2015, all major guidelines recommended treating all HIV-infected adults regardless of their CD4 count. Despite effective ART with virological suppression, HIV-associated neurocognitive disorders (HAND), cardiovascular diseases (CVD), metabolic syndrome (MS), bone abnormalities and non-HIV-associated malignancies remain a major complication associated with HIV infection. In this review article, I would like to describe recent ART status and problems in the ART-era.
Pub.: 09 Nov '16, Pinned: 28 Aug '17
Abstract: Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50) of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.
Pub.: 19 Nov '10, Pinned: 28 Aug '17
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