Timothy J. Fitzmaurice

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (T_H) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1β production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1β expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand—cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted. Innate responses to bacterial infection such as Chlamydia trachomatis activate inflammasomes to enable the processing of IL-1β, IL-18 and the induction of an inflammatory form of cell death termed pyroptosis. Inflammasomes are crucial to host defence but require tight regulation in order to prevent inappropriate inflammation and immunopathology. Here, we demonstrate that the pro-inflammatory potential of an attenuated strain of Chlamydia trachomatis, that fails to activate the inflammasome, can be rescued by the addition of a bacterial metabolite. The requirement for this metabolite, highlights a novel mechanism of inflammasome regulation and reveals a crucial role for STING mediated interferon signalling independent of cGAS. These findings further our understanding of how the innate immune system can differentiate between potential infectious and non-infectious threats and mount appropriate immune responses.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

Understanding the genetic, antigenic and structural changes that occur during HIV-1 infection in response to pre-existing immunity will facilitate current efforts to develop an HIV-1 vaccine. Much is known about HIV-1 variation at the population level but little with regard to specific changes occurring in the envelope glycoprotein within a host in response to immune pressure elicited by antibodies. The aim of this study was to track and map specific early genetic changes occurring in the viral envelope gene following vaccination using a highly controlled viral challenge setting in the SHIV macaque model. We generated 449 full-length env sequences from vaccinees, and 63 from the virus inoculum. Analysis revealed a different pattern in the distribution and frequency of mutations in the regions of the envelope gene targeted by the vaccine as well as different patterns of diversification between animals in the naïve control group and vaccinees. Given the high stringency of the model it is remarkable that we were able to identify genetic changes associated with the vaccination. This work provides insight into the characterization of breakthrough viral populations in less than fully efficacious vaccines and illustrates the value of HIV-1 Env SHIV challenge model in macaques to unravel the mechanisms driving HIV-1 envelope genetic diversity in the presence of vaccine induced-responses.

Susceptibility to scrapie disease in sheep, the archetypal prion disease, correlates with polymorphisms within the ovine PrP (prion-related protein) gene. The VRQ (Val¹³⁶Arg¹⁵⁴Gln¹⁷¹) and AL141RQ (Ala¹³⁶Leu¹⁴¹Arg¹⁵⁴Gln¹⁷¹) allelic variants are associated with classical scrapie, whereas the ARR (Ala¹³⁶Arg¹⁵⁴Arg¹⁷¹), AF141RQ (Ala¹³⁶Phe¹⁴¹Arg¹⁵⁴Gln¹⁷¹) and AHQ (Ala¹³⁶His¹⁵⁴Gln¹⁷¹) allelic variants are associated with atypical scrapie. Recent studies have suggested that there are differences in the stability of PrPSc (abnormal disease-specific conformation of PrP) associated with these different forms of scrapie. To address which structural features of ovine PrP may contribute to this difference, in the present study we have investigated the conformational stability and susceptibility to aggregation of allelic variants of ovine PrP associated with classical or atypical scrapie. We find that the melting temperature of ovine recombinant VRQ and AL141RQ PrP is higher than that of AF141RQ, AHQ and ARR. In addition, monoclonal-antibody studies show that the region around helix-1 of VRQ and AL141RQ is less accessible compared with other ovine PrP allelic variants. Furthermore, the extent of both the structural change to copper-ion-treatment and denaturant-induced aggregation was reduced in PrP associated with atypical scrapie compared with PrP associated with classical scrapie. Through the use of molecular dynamics simulations we have found that these biochemical and biophysical properties of ovine PrP correlate with the ease of unwinding of helix-2 and a concurrent conformational change of the helix-2–helix-3 loop. These results reveal significant differences in the overall stability and potential for aggregation of different allelic variants of ovine PrP and consequently have implications for the differences in stability of PrPSc associated with classical and atypical scrapie.

Ovine PBMCs (peripheral blood mononuclear cells) express PrPC [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrPC expression by PBMCs, together with plasma PrPC levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrPC expression on normal ovine PBMCs by FACS with the presence of PrPC in plasma measured by capture–detector immunoassay. FACS showed similar levels of cell-surface PrPC on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrPC showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrPC levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrPC. Homozygous VRQ sheep showed the highest plasma PrPC level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrPC levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrPC expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrPC in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrPC was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrPC.