Walking in the proteomic footprints of viral infection
The outcome of a viral infection is influenced by the balance between host defense mechanisms and virus modulation of cellular pathways. By making use of finely tuned protein interactions, viruses succeed in sabotaging intricate host processes and turning cellular systems to their own use. Although virus-host interactions have been extensively studied, our knowledge of the detailed virus-host protein interactions at the heart of these mechanisms remains quite limited. Our laboratory is interested in approaching the dynamic viral infection by tracking the localization and interactions of viral proteins in host systems as a function of infection time.
The work in our laboratory seeks to push the boundaries of current proteomic techniques by devising new tools for exploring protein interactions and placing them in defined biological contexts. We have developed a series of multidisciplinary strategies to be used in the context of viral infection, including identifying protein-protein and protein-nucleic acid interactions, quantifying subtle changes in protein interactions, defining distinct protein complexes, measuring the relative stability and the specificity of interactions, and defining the function of posttranslational modifications. The proteomic approaches employed in our laboratory add a powerful new dimension to the already considerable reach of molecular biology in querying nature. Further decreasing the time necessary to go from a live cell to an isolated protein complex, and to broaden the capability of simultaneous analysis, is a central goal of our lab. Employing new ways to look at long-standing questions is one of the strongest leitmotifs in science, giving us new opportunities to appreciate, as Darwin saw it, the "grandeur in this view of life."
We have successfully implemented our multidisciplinary methodologies to uncover the viral-host protein interactomes of a broad range of viruses, such as HCMV, HSV-1, PRV, HIV-1, WNV, and Sindbis. Our findings have led to new hypotheses as to how viruses manipulate host cellular processes, encompassing everything from chromatin remodeling to reorganization of the subcellular landscape. The richness of novel identified interactions reminds us that we are still in virtually uncharted territory in understanding the complexity of the virus-host relationship.
Nuclear sensing of viral DNA en route to innate immune signaling
The ability of mammalian cells to recognize foreign DNA is an essential step for the onset of immune responses against viruses. This recognition is accomplished by proteins called sensors, which bind viral DNA and induce cytokine secretion to alert neighboring cells and inhibit the spread of infection. Given the conceptual challenge of distinguishing two similar molecules (viral and cellular DNA), until recently, the sensing of viral DNA was thought to occur only in subcellular compartments typically devoid of host DNA (e.g., cytoplasm). However, this long-standing belief failed to fully explain how the cell detects nuclear-replicating DNA viruses. My lab’s characterization of the first identified nuclear DNA sensor, IFI16, has helped to establish the novel concept of nuclear sensing. We demonstrated that nuclear IFI16 senses nuclear-replicating herpesviruses, including HCMV and HSV-1. We also defined the viral immunoevasion mechanisms, demonstrating that HCMV inhibits IFI16 oligomerization and the propagation of immune signals, while HSV-1 targets IFI16 for degradation. The discovery that sensing can occur in the nucleus opens a new direction for research in immunity that will ultimately increase our understanding of how balanced immune responses work to maintain a healthy system and how their misregulation leads to immune disorders, cancers, and virus-induced mortality.
Spatiotemporal regulation of virus-host interactions
Protein interactions, of a stable or transient nature, underlie the majority of cellular processes. The localization, composition, and organization of protein complexes across space and time dictates their potential functions. Accordingly, eukaryotic cells spatially organize their proteomes to partition biological functions between the cytosol and specialized organelles. Viruses have evolved to overcome the challenge of replicating within this spatially-defined cellular landscape, and many aspects of their infection cycles are regulated or organized at the subcellular level. We seek to understand how spatial and temporal boundaries dictate virus-host protein interactions, and, in turn, how these interactions hinder or promote the viral infection cycle.
We developed an approach to define dynamic macromolecular interactions via a single fluorescent tag that allows us to both visualize proteins in live cells and capture their interacting partners with rapid, high-affinity immunopurifications. We found that this methodology opens new doors for almost every protein complex that we have approached, leading to novel insights into cellular processes as well as new questions. In addition, we have integrated confocal microscopy, density-based organelle fractionation, proteomics, and machine learning to define the subcellular organization of both host and viral proteomes across infection time. We applied this method to HCMV infection, which is known to cause broad structural changes to cellular organelles, and successfully profiled nearly 4,000 host and 100 viral proteins across five days of infection. This work revealed that HCMV infection causes a striking global reorganization of the host proteome to regulate organelle composition and function and generates specialized infection-induced compartments. In addition, we were able to demonstrate the spatiotemporal dynamics of previously uncharacterized viral proteins, providing novel insights into their functions and importance for viral replication. This work highlighted the importance of subcellular organization during infection time, and has prompted many ongoing follow-up studies into the dynamic, localization-dependent functions of both host and viral proteins. Continued investigation of infection-induced subcellular remodeling has also challenged us to develop new imaging and multidimensional analysis techniques to parallel the proteomic investigations of exchanges between subcellular compartments.
Sirtuins as evolutionary conserved antiviral factors and posttranslational modifiers
Most currently available antiviral drugs target specific viral proteins. Consequently, they work for only one virus and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which would provide effective targets for therapeutic treatments that would limit the evolution of viral resistance. From our virus-host protein interaction studies, we found that viral proteins target human sirtuin (SIRT) enzymes during infections with several different DNA and RNA viruses. We followed this finding with studies aimed at characterizing the functions of SIRTs. These studies have led to the discovery that the seven human SIRTs have antiviral functions against both DNA and RNA viruses. We demonstrated that SIRT-activating drugs inhibit the replication of diverse viruses, including HCMV—a slowly replicating DNA virus, and influenza H1N1—an RNA virus that multiplies rapidly. Furthermore, we demonstrated that these defense functions are evolutionarily conserved, as CobB, the SIRT homologue in E. coli, protects against bacteriophages.
In addition to their antiviral roles, SIRTs have been characterized as critical enzymes that govern genome regulation, metabolism, aging. However, the knowledge of the mechanisms involved in their multiple functions remains limited. Previous work on SIRTs suggested they may act via catalysis of posttranslational modifications, including acetylation, ribosylation, and sumoylation. However, despite having a conserved deacetylase domain, a robust catalytic activity for the mitochondrial SIRT4 remained elusive. Using a hybrid proteomics, molecular biology, and biochemistry approach, we established SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). PDH plays a central role in cellular metabolism, coupling metabolic flux from glycolysis into the TCA cycle and fatty acid synthesis. While previously thought to be primarily regulated by phosphorylation, our study helped to shift this canonical notion regarding PDH regulation. We discovered PDH as a biological substrate of mitochondrial SIRT4, demonstrating that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. Our findings define SIRT4 as an important regulatory hub of energy and lipid metabolism, which we expect to help in future studies of cellular metabolism pathways and of metabolic disorders. Furthermore, this is the first characterization of a mammalian cellular lipoamidase.
Altogether, our findings highlight SIRTs as ancient defense factors that protect against a variety of viral pathogens and exhibit diverse posttranslational modification mechanisms. Our work has provided a framework for elucidating a new set of host cell defense mechanisms and developing SIRT modulators with antiviral activity, and continues to be an exciting venue of research in our laboratory.
Proteomic-Genomic approaches to the role of chromatin remodeling complexes during viral infection
One of the intriguing findings from our virus-host interaction studies was that chromatin-remodeling enzymes are targeted by viruses, such as HCMV, HSV-1, and HIV-1. This is an exciting finding with broad implications, as it indicates that viruses hijack these enzymes to control host or viral gene expression and, in turn, the outcome of an infection. Indeed, we found that histone deacetylases (HDACs), an important class of transcriptional and epigenetic regulators, are modulated during infection. Selected HDACs have been the subject of intense study and are targets for anti-cancer therapy. However, the functions of many HDACs are not yet fully understood. We have built the first functional protein interaction network for all eleven human HDACs, and have studied their regulation by diverse posttranslational modifications. For example, we identified class IIa HDACs as novel substrates of the essential mitotic kinase Aurora B. We proposed that their sequestration within a phosphorylation gradient at the midzone contributes to a cell cycle-dependent loss of transcriptional repressive capabilities. Our work on HDACs has wide-reaching implications and has been instrumental for the understanding of gene regulation both in the context of viral infection and basic cell biology.