Periodic Reporting for period 4 - UbiProPox (Modulation of the Ubiquitin Proteasome System During Multiple Stages of the Poxvirus Lifecycle)
Période du rapport: 2020-01-01 au 2021-08-31
Vaccinia virus (VACV), the prototypic poxvirus, is a large, enveloped, DNA virus characterized by its cytoplasmic site of replication and large subset of genes. Due to the complexity of VACV, the majority of studies focus on the virus rather than the host cell. Thus, the repertoire of cell factors and functions required for its replication remain largely unknown. Our previous work to define a subset of these, revealed the cellular degradation machinery as a key requirement of VACV replication. Our findings indicated that ubiquitin (Ub), Ub ligase activity, and proteasome-mediated degradation are required for multiple stages of the virus lifecycle.
The aim of this project is to reveal how VACV differentially modulates or takes advantage of cellular degradation systems during genome uncoating, the initiation of DNA replication, and the assembly of progeny virions. For genome uncoating we will characterize the spatial and temporal interactions between ubiquitinated viral proteins, proteasomes, the viral uncoating factor, and the viral genome that occur on cytoplasmic cores. To ascertain how Cullin-3 based ubiquitination and proteasome degradation facilitate the switch from uncoating to replication of the viral genome, we will identify the relevant Cullin-3 substrates in the context of a detailed characterization of viral replication initiation sites. Coming full circle, we will explore the mechanisms used by VACV to modulate cellular degradation such that ubiquitinated viral core proteins are packaged into newly forming virions without being degraded. Using systems biology, virology, cell biology, biochemistry, molecular biology and a wide range of microscopy approaches we will unravel the complex interactions between poxviruses and the host cell degradation machinery. In turn, as viruses often serve as valuable tools to study cell function, this work is likely to uncover new insights into how cells spatially and temporally regulate their own degradative capacities.
For society, the development of new anti-viral strategies against poxviruses, and other viruses, requires detailed information of the entire replication cycle and the cellular factors and processes involved. Thus, on an applied level these studies may facilitate the identification of novel cellular anti-viral targets with broad-spectrum antiviral potential.
The aim of this project is to reveal how VACV differentially modulates or takes advantage of cellular degradation systems during genome uncoating, the initiation of DNA replication, and the assembly of progeny virions. For genome uncoating we will characterize the spatial and temporal interactions between ubiquitinated viral proteins, proteasomes, the viral uncoating factor, and the viral genome that occur on cytoplasmic cores. To ascertain how Cullin-3 based ubiquitination and proteasome degradation facilitate the switch from uncoating to replication of the viral genome, we will identify the relevant Cullin-3 substrates in the context of a detailed characterization of viral replication initiation sites. Coming full circle, we will explore the mechanisms used by VACV to modulate cellular degradation such that ubiquitinated viral core proteins are packaged into newly forming virions without being degraded. Using systems biology, virology, cell biology, biochemistry, molecular biology and a wide range of microscopy approaches we will unravel the complex interactions between poxviruses and the host cell degradation machinery. In turn, as viruses often serve as valuable tools to study cell function, this work is likely to uncover new insights into how cells spatially and temporally regulate their own degradative capacities.
For society, the development of new anti-viral strategies against poxviruses, and other viruses, requires detailed information of the entire replication cycle and the cellular factors and processes involved. Thus, on an applied level these studies may facilitate the identification of novel cellular anti-viral targets with broad-spectrum antiviral potential.
My lab has focused on determining the complex interactions between viral proteins needed for genome release and replication and the cellular factors we have identified as necessary for theses processes. We have investigated biochemical and/or genetic relationships between viral encoded enzymes (ATPases, kinases and phosphatases) and the cells protein disposal machinery. Through this we have identified a link between viral kinases and cellular degradation activity. We're now focused on determining the role of these kinases in controlling host cell degradative functions and how this impacts the virus’s ability to replicate.
During this project, we made major advances in our understanding of how VACV subjugates cellular degradation machineries to facilitate its infectious cycle. We have investigated the role of ubiquitin and di-ubiquitin (sumo) in the regulation of virus entry, and uncovered essential roles for cellular stress proteins and cellular degradative membrane compartments in virus entry assembly. For the cell stress proteins, termed heat shock proteins (HSPs) we found that they are required for virus genome uncoating and virus assembly. Given their ability to block two major stages of virus replication, we will pursue HSP inhibitors as potential anti-viral agents in the future.
We have made progress in characterizing the spatial/temporal relationship of cellular degradation machinery and sites of virus replication using super-resolution microscopy. We have determined which waste tags are subjugated by the virus to help it infect host cells. We have also investigated the spatial/temporal patterning of sumo, a di-ubiquitin molecule, during VACV infection. We have identified major changes in its distribution during infection and have revealed that several viral proteins harbor strong sumo acceptor sites. We also have evidence that sumoylated viral proteins are packaged into forming virions, a novel and exciting finding.
For the cell stress proteins, termed heat shock proteins (HSPs) we found that they are required for virus genome uncoating and virus assembly. Given their ability to block two major stages of virus replication, we will pursue HSP inhibitors as potential anti-viral agents in the future.
Regarding cellular degradative membrane compartments, we found that multivesicular bodies – late-stage endocytic degradative compartments - are stolen by the virus and used in the process of virus wrapping and egress. This finding not only expands our understanding of poxvirus biology but opens many new questions regarding how the viruses hijacks these compartments on a mechanistic level, the impact of virus-mediated release of MVBs on host immunity and the potential therapeutic effect of targeting this pathway to block virus release and the spread of infection in a host organism.
These findings have been disseminated through presentations to the scientific community at national and international scientific meetings and througn publication of these results in scientific journals. Dissemination of the work, and its implications, through social media and presentations has been used to engage lay audiences with this work.
During this project, we made major advances in our understanding of how VACV subjugates cellular degradation machineries to facilitate its infectious cycle. We have investigated the role of ubiquitin and di-ubiquitin (sumo) in the regulation of virus entry, and uncovered essential roles for cellular stress proteins and cellular degradative membrane compartments in virus entry assembly. For the cell stress proteins, termed heat shock proteins (HSPs) we found that they are required for virus genome uncoating and virus assembly. Given their ability to block two major stages of virus replication, we will pursue HSP inhibitors as potential anti-viral agents in the future.
We have made progress in characterizing the spatial/temporal relationship of cellular degradation machinery and sites of virus replication using super-resolution microscopy. We have determined which waste tags are subjugated by the virus to help it infect host cells. We have also investigated the spatial/temporal patterning of sumo, a di-ubiquitin molecule, during VACV infection. We have identified major changes in its distribution during infection and have revealed that several viral proteins harbor strong sumo acceptor sites. We also have evidence that sumoylated viral proteins are packaged into forming virions, a novel and exciting finding.
For the cell stress proteins, termed heat shock proteins (HSPs) we found that they are required for virus genome uncoating and virus assembly. Given their ability to block two major stages of virus replication, we will pursue HSP inhibitors as potential anti-viral agents in the future.
Regarding cellular degradative membrane compartments, we found that multivesicular bodies – late-stage endocytic degradative compartments - are stolen by the virus and used in the process of virus wrapping and egress. This finding not only expands our understanding of poxvirus biology but opens many new questions regarding how the viruses hijacks these compartments on a mechanistic level, the impact of virus-mediated release of MVBs on host immunity and the potential therapeutic effect of targeting this pathway to block virus release and the spread of infection in a host organism.
These findings have been disseminated through presentations to the scientific community at national and international scientific meetings and througn publication of these results in scientific journals. Dissemination of the work, and its implications, through social media and presentations has been used to engage lay audiences with this work.
In addition to the biology-oriented findings of this project, using the virus as a tool we have also developed methods for the mapping multi-protein structures using super-resolution microscopy techniques. While initially applied to the virus, these tools are freely available and applicable to a wide range of pathogens as well as complex cellular structures. We have also made technological advances using a combination of machine learning and Artificial Intelligence to develop novel automated methods for detecting viruses and changes in virus infection patterns in 3-D and 4-D, at the single cell and population level. These analysis tools are beyond the state-of-the-art in the field. We have developed machine learning and artificial intelligence-based analysis pipelines that allow us to extract information and classify phenotypes that were previously overlooked. These developments have been used to analyze very large complex datasets of VACV uncoating, replication and assembly. These tools are freely available and will be useful for the analysis of other viruses and complex cellular biological processes. These tools have already contributed to two related host pathogen studies. In the future we will look to expand these analysis tools to even more pathogens and cell systems with diagnostic and clinical applications in mind.