Doctor of Philosophy

dissertationMammalian hosts have evolved protein "restriction factors" to combat retroviruses. TRIM5α and the related TRIMCyp protein (collectively TRIM5), are restriction factors that can potently restrict retroviruses, including HIV-1, by binding their capsids and blocking reverse transcription. Previous studies have shown that the C-terminal SPRY/CypA domains of TRIM5 proteins bind the capsids of susceptible retroviruses and that higher-order oligomerization of TRIM5 proteins apparently also contributes to capsid binding. However, the biochemical and structural details of these interactions are not fully understood. To study how TRIM5 proteins recognize capsids, we have developed new methods for expressing and purifying recombinant TRIM5 proteins. Here, we report biochemical, electron microscopic and X-ray crystallographic studies of pure recombinant TRIM5 proteins and their complexes with authentic HIV-1 core particles and in vitro-assembled mimics of the HIV-1 capsid surface. In Chapter 2, we report the expression, purification and electron crystallographic studies of a restrictive, but non-native chimeric rhesus TRIM5 protein (TRIM5-21R), and show that TRIM5-21R can spontaneously self-assemble into paracrystalline hexagonal lattices comprising 6-sided rings. Moreover, ring assembly is promoted by TRIM5-21R binding to hexagonal HIV-1 CA assemblies. In Chapter 3, we report the first crystal structure of a TRIM coiled-coil domain (from human TRIM25) as well as supporting analytical ultracentrifugation and disulfide crosslinking experiments showing that other iv TRIM coiled-coils, including TRIM5, also form antiparallel dimers that are ~170 Å long. In Chapter 4, we describe the expression and purification of 11 different mammalian TRIM5 alleles. We demonstrate that TRIM5 hexagonal assembly is a conserved property and report electron microscopic and biochemical studies showing that TRIM5 proteins form a ~35 nm-spaced, flexible hexagonal "net" on the surface of decorated HIV-1 cores and other capsid mimics. In Chapter 5, I present my ongoing attempts to crystallize and determine the structure of the TRIM5α core domains in the assembled state. Taken together, my work supports a "pattern recognition" model for capsid recognition in which TRIM5 proteins have evolved to restrict a variety of different retroviruses by cooperatively assembling flexible hexagonal nets that can bind avidly and adapt to the symmetry, hexagonal spacing and curvature of retroviral capsids

Decoding the Distribution of Glycan Receptors for Human-Adapted Influenza A Viruses in Ferret Respiratory Tract

Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface. It is generally known that upper respiratory tract of humans and ferrets predominantly express α2→6 sialylated glycan receptors. However much less is known about the fine structure of these glycan receptors and their distribution in different regions of the ferret respiratory tract. In this study, we characterize distribution of glycan receptors going beyond terminal sialic acid linkage in the cranial and caudal regions of the ferret trachea (upper respiratory tract) and lung hilar region (lower respiratory tract) by multiplexing use of various plant lectins and human-adapted HAs to stain these tissue sections. Our findings show that the sialylated glycan receptors recognized by human-adapted HAs are predominantly distributed in submucosal gland of lung hilar region as a part of O-linked glycans. Our study has implications in understanding influenza A viral pathogenesis in ferrets and also in employing ferrets as animal models for developing therapeutic strategies against influenza.Singapore-MIT Alliance for Research and Technolog

Visualizing formation of the active site in the mitochondrial ribosome.

Funder: Agouron InstituteFunder: Louis-Jeantet FoundationRibosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis

Phenotypic and genotypic characterization of B.t.LDC-391 strain that produce cytocidal proteins against human cancer cells

An indigenous Bacillus thuringiensis strain B.t.LDC-391 producing cytocidal proteins against human colon cancer cell line, HCT-116, was subjected to phenotypic and genotypic characterization to evaluate its relatedness to B.anthracis. The morphological features of this strain were meta-analyzed with data of other parasporin and insecticidal protein producing Bacillus thuringiensis strains. The conventional biochemical analysis and antibiotic sensitivity test proved it as an ampicillin resistant which is a salient feature, absent in B.anthracis Ames. PCR analysis showed the absence of cyt and parasporin related genes in the genome of B.t.LDC-391. But the strain was positive for cap gene. The sequencing and bio-informatic analysis of cap gene and 16S rDNA of B.t.LDC-391 placed it closer to B.thuringiensis and revealed significant divergence from that of any B.anthracis strain. However our strain lacked β– hemolysis on human erythrocytes which is a common feature of B.anthracis strains and parasporin producers

Glycomics-based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses.Singapore-MIT Alliance for Research and Technolog

Physical activity patterns and gestational diabetes outcomes – The wings project

AbstractObjectiveTo compare physical activity (PA) patterns in pregnant woman with and without gestational diabetes (GDM) and to assess the effects of an exercise intervention on change in PA patterns, blood glucose levels and pregnancy outcomes in GDM women.MethodsFor the first objective, PA patterns were studied in 795 pregnant women with and without GDM. For the second objective, the Women in India with Gestational Diabetes Strategy-Model of Care (WINGS-MOC) intervention were evaluated in 151 women out of 189 with GDM. PA was assessed using a validated questionnaire and a pedometer. Changes in PA patterns, glycemic parameters and neonatal outcomes were evaluated.ResultsOverall, only 10% of pregnant women performed recommended levels of PA. Women with GDM were significantly more sedentary compared to those without GDM (86.2 vs. 61.2%, p<0.001). After the MOC was implemented in women with GDM, there was a significant improvement in PA and a decrease in sedentary behaviour amongst women (before MOC, moderate activity: 15.2%, sedentary: 84.8% vs. after MOC-moderate: 26.5%, sedentary: 73.5%; p<0.001), and an increase in their daily step count from 2206/day to 2476/day (p<0.001). Fasting 1 and 2-h postprandial glucose values significantly decreased (p<0.001 for all). Sedentary behaviour was associated with a fourfold higher risk (p=0.02), and recreational walking with 70% decreased risk, of adverse neonatal outcomes (p=0.04) after adjusting for potential confounders.ConclusionsPA levels are inadequate amongst this group of pregnant women studied i.e. those with and without GDM. However, a low-cost, culturally appropriate MOC can bring about significant improvements in PA in women with GDM. These changes are associated with improved glycemic control and reduction in adverse neonatal outcomes

Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.National Institute of General Medical Sciences (U.S.) (GM57073)National Institute of General Medical Sciences (U.S.) (U54 GM62116)Singapore. Agency for Science, Technology and ResearchSingapore-MIT Alliance for Research and Technolog

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.National Institute of General Medical Sciences (U.S.) (Glue Grant U54 GM62116)National Institutes of Health (U.S.) (Grant GM57073)Singapore-MIT Alliance for Research and Technolog