Cristea Lab Research
The research of our laboratory is at the interface between virology and proteomics. We are studying cellular mechanisms used in defense against viruses, as well as mechanisms used by viruses to inhibit or hijack host cell processes. For these studies, we use multidisciplinary approaches, integrating molecular virology, microscopy, mass spectrometry-based proteomics, and bioinformatics. We have developed a series of proteomics-based approaches for characterizing cellular processes occurring during the progression of viral infections, and we are exploring several areas of interest:
- Dynamic regulation of host-virus protein-protein and protein-nucleic acid interactions during the progression of an infection.
- Mechanisms of sensing of pathogenic DNA within the nuclei of infected cells
- Initiation and propagation of intrinsic and innate immune responses following infection with nuclear-replicating herpesviruses
- Roles and regulation of human deacetylases (histone deacetylases - HDACs and sirtuins - SIRTs) during viral infections
- Global remodeling of cellular organelles during the progression of viral infection
- Developing targeted and large-scale proteomic tools for quantifying proteins, identifying protein interactions, determining the specificity of interactions, defining distinct protein complexes, and measuring the relative stability of interactions
Protein interactions, of a stable or transient nature, underlie the majority of cellular processes. Knowledge of the localization, composition, and organization of protein complexes at a particular point in space and time provides key insights into their functions. We have developed a strategy that uses a single fluorescent tag to both visualize proteins in live cells and capture their interacting macromolecular partners via high-affinity immunopurifications. My previous work showed that rapid isolations are essential to preserving protein complexes close to their original state in the cell. Accordingly, we use cryogenic cell lysis in combination with 5-60 minutes immunoisolations on magnetic beads coated with in-house developed antibodies. We found that this methodology opens new doors for almost every protein complex that we have approached, leading to both insights into cellular processes and new questions. However, gaining access to low-stoichiometry and short-lived interactions is still challenging, and further methodological improvements are a necessity. The work in my laboratory will seek to push the boundaries of current proteomic techniques, and continue to devise new tools for exploring protein interactions. Effort will be placed into further decreasing the time necessary to go from a live cell to an isolated protein complex.
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 interactions between viral and host proteins during the course of infection remains quite limited. I was 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. We have successfully implemented our new methodologies to uncover the viral-host protein interactomes of the mosquito-borne Alphavirus Sindbis, HIV-1, and, most recently, the human cytomegalovirus. While our findings led to new hypotheses as to how viruses manipulate host cellular processes, the richness of the novel identified interactions reminded us that we are still in virtually uncharted territory in understanding the complexity of the viral-host protein relationship.
The research in my laboratory is aimed at understanding mechanisms that control the fate of cells under invasion by pathogens, with particular emphasis on the viral modulation of chromatin remodeling machineries. The wide range of diseases caused by viruses, from the common cold to arthritis to AIDS, is a reflection of the diverse ways in which viruses interact with host cells and manipulate their basic functions. How do viruses accomplish these tasks, and how do cells respond and adapt to infection? In approaching this question, we use the full power of differential genomics and proteomics to study the modulation of chromatin remodeling complexes in both normal and virally infected cells. The state-of-the-art mass spectrometry in my laboratory focuses on the use of novel instrument configurations, which are ideally suited for the fast and sensitive characterization of macromolecular complexes.
In all eukaryotes, gene expression is regulated by chromatin remodeling complexes, which use an intricate system of specialized enzymes to coordinate a series of posttranslational modifications on the N-terminal tails of histones. Our preliminary studies indicated that viruses may modulate gene expression through interactions with chromatin remodeling complexes. Indeed, histone deacetylases are targeted during infection by numerous viruses, and are reported to mediate the latency stages of cells infected with HSV-1 and HIV. Our approach provides a close look at this relationship, showing that HIV, HCMV, and Sindbis viral proteins recruit members of the chromatin remodeling complexes, including histone deacetylases, acetyltransferases, demethylases, and methyltransferases, as well as histone chaperones. While such interactions are in part virus-specific, some may reveal common pathways utilized by viruses to determine the outcome of the infection or by the cell in response to injury.
Characterizing chromatin remodeling complexes in normal and virally infected cells. Now that we have robust and efficient methodologies for the study of macromolecular interactions, we have initiated a comprehensive mapping of the chromatin remodeling interactome in normal cells. The novel interacting partners of HDAC1 so far uncovered are indicative of the multi-faceted roles of HDAC complexes in the precise orchestration of histone modifications, and we are currently exploring the roles of these specific interactions. Additionally, these analyses of unperturbed cells provide a baseline for further studies of the way cells respond to changing conditions. In particular, we will examine the effects of viral infection on chromatin remodeling complexes using approaches such as 1) tracking the localization of members of the chromatin remodeling complexes, 2) revealing the proteins that assemble these complexes, 3) identifying changes in protein-DNA interactions, 4) mapping posttranslational modifications on chromatin remodeling complexes and their targets, 5) determining protein turnover using mass spectrometry, and 6) elucidating the structures of chromatin remodeling assemblies using proteomics-based methodologies.
The proteomic approaches employed in our laboratory add a powerful new dimension to the already considerable reach of molecular biology in querying nature. 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."