Mak Group

Supervisors and collaborators: Prof Johnson Mak, Mr Erwan Bremaud, and Dr Belinda de Villiers

Virology and Cell Biology

Polarized or precision targeting of protein complexes to their destinations is fundamental to cellular homeostasis, but the mechanism underpinning directional protein delivery is poorly understood. Using the uropod targeting HIV synapse as a model system, we have shown that the viral assembly machinery Gag is co-polarized with the intracellular calcium (Ca²⁺) gradient and binds specifically with Ca²⁺. Conserved glutamic/aspartic acids flanking endosomal sorting complexes required for transport binding motifs are major Ca²⁺ binding sites. Deletion or mutation of these Ca²⁺ binding residues resulted in altered protein trafficking phenotypes, including (i) changes in the Ca²⁺–Gag distribution relationship during uropod targeting and/or (ii) defects in homo/hetero-oligomerization with Gag. Mutation of Ca²⁺ binding amino acids is associated with enhanced ubiquitination and a decline in virion release via uropod protein complex delivery. Our data that show Ca²⁺–protein binding, via the intracellular Ca²⁺ gradient, represents a mechanism that regulates intracellular protein trafficking. Delineating the polarised protein trafficking underpinning distinct biological processes could have broad implication in HIV transmission.

Techniques: Molecular Virology, Protein Biochemistry, Protein Biophysics, Imaging Analyses, and Primary Cell Organoids Systems.

Supervisors: Dr Thomas Ve, Dr Yun Shi, Dr Andrea Maggioni, Prof Johnson Mak & Prof Mark von Itzstein

Structural Biology, Biochemistry, Virology

Nipah virus (NiV) is a highly lethal (risk group 4) zoonotic paramyxovirus causing severe, rapidly progressive encephalitis in humans with the case fatality rate ranging from 40-70%. NiV is closely related to Hendra virus (HeV), another risk group 4 paramyxovirus that is native to Australia and infects both horses and humans. NiV is widely distributed in Southeast Asia, India, and Africa. WHO has earmarked NiV on a priority list of eight pathogens that is expected to cause severe outbreaks in the near future. While a one-health approach of vaccinating the intermediate host (horse in the case of Hendra) is able to block the transmission of Hendra virus, the limited uptake of the Hendra vaccine by horse owners could potentially make such approach ineffective. Furthermore, transmission of NiV to humans may occur after direct contact with infected bats, infected pigs, or from other NiV infected people making a one-health preventive approach not practical to NiV, due to the lack of reliance of an intermediate host.

The NiV envelope proteins, glycoprotein G and fusion protein F, are the determinants of viral entry. G and F achieve this via their recognition of the host- cell surface proteins Ephrin-B2 and -B3, and the glycosaminoglycan heparan sulfate (HS). Although detailed structural information is available for the G/Ephrin-B2/B3 interactions, the structural basis for how the G protein coordinates selective binding to specific glycans, such as HS is completely unknown. Furthermore, the mechanistic details of how any of the host-cell receptors trigger viral fusion are poorly defined.

This project will involve a combination of biophysical and cell-biology approaches and aims to define the molecular basis of NiV/HeV interaction with host-cell glycans (glycointeractome), define the molecular mechanisms underlying fusion activation and identify inhibitors targeting these processes. A student working on this project may specialise in one particular aspect or be involved with a number of the different aspects of the project.

Techniques: X-ray crystallography, cryo electron microscopy (cryo-EM), saturation transfer difference nuclear magnetic resonance (STD NMR), library screening, surface plasmon resonance (SPR), glycan arrays, and viral infection assays using pseudotyped particles.

Supervisors: Dr Belinda de Villiers and Prof Johnson Mak

Both the speed of rapid antigen test (RAT) and the accuracy of laboratory (lab)-based polymerase chain reaction (PCR) are critical in managing infectious diseases. Early diagnoses to discern clinically indistinguishable viruses that require specific/precision treatment options are invaluable. Clustered regularly interspaced short palindromic repeats-diagnostic (CRISPR-Dx) combines the speed of RAT and the accuracy of PCR. Using CRISPR-associated protein 13a (Cas13a) in CRISPR-Dx, we have detected and quantified highly related viral RNAs across variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our SARSCoV-2 CRISPR-Dx is precise, sensitive (within low femtomolar), and rapid (< 1hr). Despite its accuracy and speed, the low conversion rate of synthetic CRISPR-Dx reagents (i.e., from design to verification of pathogen specific single guide RNAs [sgRNA]) limits broad adaptation of CRISPR-Dx as a mainstream diagnostic. The bottleneck in robust identification of sgRNAs rests on the ill-defined biochemical- and biophysical-dynamics amongst a) sgRNA; b) target RNA; and c) Cas13a protein. Furthermore, the inter-relationships across sgRNA, target RNA, and Cas13 enzyme are multi-factorial and contextual (hierarchical and non-linear), making it challenging to assure any bioinformatic design will function as potent sgRNAs 1. This hurdle can be bypassed using validated artificial intelligence (A.I.) pipelines to improve the design of functional sgRNAs for CRISPR-Dx. A robust CRISPR-Dx platform will transform our preparedness in managing infectious diseases, we will leverage our diverse structural-functional CRISPR-Dx datasets to (iii) develop generative pre-trained transformer (GPT)-based A.I. pipelines to improve sgRNAs design to safeguard future pandemic viruses.

Techniques: Virology, Biochemistry, Biophysics, Molecular Biology, Structural Biology, Glycan Biology, Animal Works

Techniques: Virology, Biochemistry, Biophysics, Molecular Biology, Structural Biology

Supervisors: Dr Belinda de Villiers and Prof Johnson Mak

Trauma and/or inflammation as mechanisms cannot fully account for the different HIV transmission rates in the presence of specific genital microbes. We have recently shown that HIV glycan shield double-up as an attachment factor via a novel ‘non-electrostatic and specific’ oligomannose (oligoman)-based interaction, offering a general mechanism to account for the opposite HIV transmission phenotypes seen in the presence of lactobacillus vs sexual transmission infection bacteria, such as Neisseria gonorrhoeae. This project will determine whether this manipulatable oligoman-based interaction has: (i) helped trapped HIV onto lactobacillus to reduce HIV transmission; and (ii) allowed HIV to hijack Neisseria gonorrhoeae to gain access into the host. This project is built upon our novel discovery that a ‘non-electrostatic, specific, manipulatable, sugar-sugar’ is an attachment factor for HIV. This interaction occurs between HIV oligomannose (oligoman) and cellular N-acetylglucosamine (GlcNAc) prior to receptor engagement. The conservation of glycan biology across domains of life and the non-living (viruses) has led to our current proposal to investigate whether this oligoman-GlcNAc (or oligoman-based) interaction may underpin some of the dynamic- interplays amongst: (i) vaginal microbes; (ii) HIV; and (iii) their human host. Our findings that HIV double up to use its oligoman enriched HIV envelope (Env) glycan shield as an attachment factor may offer a general principle to account, at least in part, for: (i) how vaginal lactobacillus offers protection from HIV transmission; and (ii) how vaginal Neisseria gonorrhoeae increases the risk of HIV transmission.

Techniques:  Virology, Biochemistry, Biophysics, Cell Biology, Molecular Biology, Glycan Biology, Structural Biology.

Supervisors: Dr Belinda de Villiers, Associate Prof Danielle Stanisic, Prof Alan Liew, and Prof Johnson Mak

Animal blood compartment is one of the most accessible organs for clinical diagnosis and prognosis. Complete blood count (CBC) is a standard operational procedure for clinicians to gain an insight on the overall health of a patient, a procedure that is informative in the prognosis of anaemia, infection, and leukemia. CBC (or blood work) is generally carried out in hospital environment and / or accredited laboratory where advance diagnostic machineries are maintained and available for testing. However, these traditional constrains are not practical for field-based or mobile clinics where sophisticated equipment are limited. Through the advancement of smart-phone and mobile devices, it is conceivable that aspects of gold-standard clinical laboratory-based CBC test can be accomplished using a combination of: (i) cloud-based software; (ii) mobile device; and (iii) purposely built biotechnology. This project will involve a combination of multi-disciplinary science, including infection diseases, software development, clinical grade sample handling, and artificial intelligence science. A student working on this project may specialise in one or multiple aspects of the project in accordance with both interest and capacity.

Techniques: Infectious Diseases, Clinical and Ex Vivo Samples Processing, Microscopy, Software Development, and Artificial Intelligence Assisted Digital Data Analyses