Agenda

In addition to well-known human pathogens such as West Nile virus, dengue virus, and yellow fever virus, the flavivirus genus includes dozens of largely unstudied viruses that circulate between animals and arthropod vectors in nature (with presumably many more yet to be discovered). Zika virus is the most recent example of an obscure arbovirus emerging as an important human pathogen. The 2015/2016 Zika virus epidemic in Latin America and the Caribbean featured unusual disease presentations including neurological disease, sexual transmission, and congenital infection. The ability of Zika virus infection during pregnancy to produce neurodevelopmental defects was unexpected because other flaviviruses do not cause birth defects and this effect had not been noted in earlier Zika virus outbreaks. I will provide an update on the ongoing Zika virus epidemic in the Americas and recent discoveries about the pathogenic mechanisms of Zika virus, as well as discuss potential future emerging flaviviruses.

Zika virus emerged in 2015-2016 in the Americas with rapid spread and substantial impact. We solved the structure of this flavivirus virion to near atomic resolution and this revealed the similarity with previous flavivirus structures such as dengue virus and West Nile virus. Despite strong overall structural similarity, regions of the envelope (E) glycoprotein revealed differences that suggested sequences that might be involved in the unique cell and tissue tropism that Zika has displayed. Together with dengue virus, we have continued to probe the structure and composition of these viruses and their assembly intermediates using the tools of cryo-electron microscopy and mass spectrometry. In particular, we have probed the process of virus particle maturation examining the immature virus particle and the conversion of prM by cellular furin to the mature M form of the protein. This process in dengue virus is very inefficient and we have been determining the stoichiometry of prM/M proteins in the Zika virion and evaluating the implications of the maturation state on virus biology. Antibody binding to the particle has also been examined and may reveal insights into the process of virus maturation as well as entry of the virion into target host cells. The ability to compare virus particles from multiple members of the flavivirus genus in terms of structure and function serves as a powerful strategy to discern common processes and distinct features that contribute to virus biology.

Studying how viruses interact with their hosts has provided us a wealth of knowledge about virus-host biology. These studies have historically relied on traditional reductionist approaches that focus on a pre-determined small set of host factors. Although these approaches have yielded notable insights into virus-host interplay, they do not provide a holistic view of the intricate relationship that viruses have with their hosts. The advent of systems biology approaches has circumvented these limitations and offered an unprecedented opportunity for global analyses of virus-host interactions. I will present two stories from our laboratory to highlight how we have harnessed the power of systems biology to answer important virological questions. The first story is about an overexpression screen that helped us identify a protein, SEC14L2, as a critical host factor for hepatitis C virus (HCV) replication. This discovery enabled us for the first time to grow HCV clinical isolates in cell culture. The second story is about an unbiased proteomics method that we have adapted to globally survey proteins targeted for cleavage in virus-infected cells. By applying this method to a panel of picornaviruses, we have identified known and several novel proteins commonly or uniquely targeted by these viruses. We have now begun to extend this method to additional viruses of human health importance, including HCV. These experiments will provide unparalleled insights into how viruses use proteolysis to remodel their host cells and create a favorable environment for their replication. Knowledge gained can then be used to improve antiviral strategies.

Chronic viral infection of the liver is a global health problem, with over 500 million individuals infected with hepatitis C or B viruses (HCV/HBV) that cause liver disease that can progress to hepatocellular carcinoma (HCC): a metastatic cancer with limited therapeutic options. Low oxygen environments, naturally found in the liver, enhance viral replication and this is mediated by hypoxia inducible transcription factors (HIFs) regulating host pathways that are essential for viral replication. Autotaxin (ATX) is a phospholipase with diverse roles in physiological and pathological processes including inflammation and oncogenesis. Clinical studies show increased ATX expression in chronic viral hepatitis, however, the pathways regulating ATX and its role in the viral life cycle are poorly understood. We demonstrate that low oxygen regulates ATX mRNA levels and hypoxia gene signature analysis of viral associated-HCC show a significant association between ATX expression levels and progressive HCC phenotype. Importantly, inhibiting ATX-lysophosphatidic acid signalling reduced HBV and HCV replication, highlighting a positive role for this phospholipase in the viral life cycle. HIFs direct extensive transcriptional responses that enable a cell to respond to diverse physiological or pathophysiological signals. Hepatitis B and C viruses activate HIFs and promote a ‘pseudohypoxic’ state. Current HCC therapies show limited efficacy and recent research efforts have focused on the molecular profiling of tumours to identify gene signatures that predict disease outcome and response to therapy. Understanding how viruses influence host gene transcription is fundamental and profiling HIF-transcriptional targets in infected cells provides a unique opportunity to define the role of HIF in viral-associated HCC heterogeneity and inform stratified approaches for new treatments.

Diversity in the mammalian immune system is essential for protecting the host against a broad range of threats, and is most clearly evident during dynamic processes such as differentiation and antigenic response. Recent years have witnessed transformative and intersecting advances in nanofabrication and molecular biology that enable deep profiling of low-input samples. Collectively, these afford new and exciting opportunities to study heterogeneity in immune responses, starting from the level of the single cell, with the potential to fundamentally advance our understanding of systems-level immune regulation in health and disease. Illustratively, I will discuss how we can leverage single-cell genomic approaches – and, in particular, single-cell RNA-Seq – to explore the extensive functional diversity among immune cells, and uncover, from the bottom-up, distinct cell states and their molecular drivers. Finally, I will discuss emerging experimental strategies for achieving the statistical power and control necessary to reconstruct intra- and inter-cellular circuits, enumerate and redefine cell states and types, and transform our understanding of systems-level cellular decision-making on a genomic scale.

There are currently over 36 million people in the world living with HIV. The development of a safe and effective prophylactic HIV vaccine is therefore a critical biomedical research priority. However, HIV poses unique challenges for vaccine development, including vast sequence heterogeneity as well as the early establishment of viral latency. I will outline current strategies to address these challenges, including the use of bioinformatically optimized mosaic antigens and vaccine platforms that induce robust polyfunctional antibody responses. I will discuss recent preclinical and clinical data with our mosaic Ad26/Env HIV vaccine and outline considerations for advanced clinical development of this vaccine candidate.

HCV is a major killer worldwide, and continues to spread. Curative treatments are transformative, but not a panacea. The study of HCV has revealed previously-unrecognized and broadly-applicable cell biology, innate and adaptive immune mechanisms, and pharmacologic innovations. In addition to the intrinsic value of fundamental research, HCV provides unparalleled opportunity to study human biology, successful and failed immune responses, and viral phylodynamics at multiple scales. In practical terms, ending the HCV epidemic requires improved (scalable) tools for diagnosis, management, and prevention of HCV infection. No major epidemic has been controlled without a vaccine, and there are ample reasons for this rule to apply to HCV - considering that the people most affected tend to have the least access to care.

We are about midway between the first report of a compound directly inhibiting HCV in vivo and the WHO deadline for elimination of HCV infection in 2030. There have been important advances including development of point of care tests to detect exposure to HCV infection, elimination of transfusion transmission of infection where testing is deployed, and of course, production of pangenotypic, safe treatments that eliminate HCV infection in nearly everyone who can afford them. HCV infection has already been eradicated from many patients, and several populations are on track for elimination. In the Netherlands, treatment has already been shown to reduce transmission. There also remain enormous challenges. Most of those infected around the world are not aware of their status. Confirmation of HCV antibodies cannot be done routinely at the point of care. Many of those with chronic infection have no access to efficacious safe medications and by the time some are cured they remain at increased risk for liver cancer. Resurgence of new infections in some areas like the United States also underscores the anemic support for HCV vaccine development and, more than 25 years later, the absence of a vaccine to prevent infection. In principle, HCV elimination goals can be achieved. Important lessons are rapidly emerging from programs in Australia, the Republic of Georgia, India, and several EU countries. With measures to promote testing and treatment uptake, a public health approach to HCV is possible. Even in the epicenter of HCV (Egypt) and even among the most difficult to treat (e. g., persons who inject drugs in India) there is substantial progress. Clearly expanded global commitment is needed to expand testing and treatment, finish vaccine development, and eliminate HCV by 2030.