Trevor Glaros (Presenter)
Edgewood Chemical Biological Center
Bio: Dr. Trevor Glaros got his undergraduate in microbiology at Clemson University and received his Ph.D. in Biology from Virginia Tech. He is a principal investigator and director of the mass spectrometry core facility at the U.S. Army’s Edgewood Chemical Biological Center in Edgewood, MD. Dr. Glaros is the lead scientist for several different studies at ECBC which collectively aim to discover new biomarkers or detection methods for biological or chemical agents.
Authorship: Andy Kilianski(1), Amanda Piper(2), Ricardo Vancini(2), Raquel Hernandez(2), Trevor Glaros(1)
(1) BioSciences Division, BioDefense Branch, Edgewood Chemical Biological Center, Gunpowder, MD 21010; (2) Department of Molecular and Structural Biochemistry, North Carolina State University, Ralei
| Short Abstract As virus particles bud from an infected cell, they contain not only virus-specific protein and nucleic acid, but they also incorporate host proteins. While this phenomenon has been observed for a handful of viruses, it remains unclear if host proteins are specifically packaged and if they are critical to the viral life cycle. In this study, the protein content of a prototypical alphavirus, Sindbis virus, was analyzed by liquid chromatography mass spectrometry to determine if host proteins are specifically packaged by these viruses and if these host proteins are critical for the viral life cycle. |
Long Abstract
Introduction:
The continuing emergence of novel and re-emergent viral pathogens creates risks to public health and endangers our deployed warfighters. To prevent and respond to these challenges, the biodefense community must develop new methods and technology for pathogen characterization. Detailing the composition of viral particles, including the host protein content, will aid in the design of new detection assays, countermeasures, and forensic techniques. Two families of viruses, Alphaviridae and Flaviviridae, are especially capable of threatening the warfighter. Notable members of these families include Chikungunya virus, Dengue virus, West Nile virus, Zika virus, and the biodefense-relevant alphaviruses Venezuelan Equine Encephalitis virus and Eastern Equine Encephalitis virus. Many of these pathogens originate from zoonotic reservoirs and lack effective vaccines or antivirals. Host-based therapeutics are thought to represent an attractive avenue, but identifying targets for this approach requires a detailed understanding of how these viruses interact with host factors throughout the replication cycle. In addition to being attractive countermeasure targets, host proteins give potential signatures related to pathogen growth conditions, background, and location. These signatures can be used in forensic applications when searching for information related to pathogen attribution. Central to either of these applications is the necessity to understand the interactions between viral pathogens and their host. To uncover these types of interactions, this research project aims first to identify which host proteins are present within the prototypical alphavirus, Sindbis virus (SINV). Then, by examining another host species and re-examining the virion’s protein content, we can determine the degree conservation of host signatures.
Methods:
For this work, SINV was grown in BHK21 (hamster), HEK293 (human), and C710 cells (mosquito). Culture medium from each cell line, infected and uninfected negative control, was harvested for a two-step viral purification procedure. First, the virus was crudely isolated from other culture media components by density ultracentrifugation. Bands corresponding to the viral particles were then collected, washed, and purified using gravity filtration columns. The final highly purified preparations were quantified for total protein content using the bicinchoninic acid assay and normalized for sample processing. Viral preparations and their respective negative controls were processed for LC-MS/MS analysis using the filter-aided sample preparation (FASP) method as previously described.1 Processed protein samples were digested with trypsin and desalted using C18 spin columns prior to mass spectrometry analysis. Tryptic peptides were analyzed on an Orbitrap Velos ELITE mass spectrometer coupled with the Easy-nLC II liquid chromatography pump system. Dried peptides from each sample were resolved on virgin 15cm HPLC columns packed with 5µm BioBasic C18 particles 300Å using a 130 minute multistep gradient. By using a new column for each sample we ensured no sample to sample carry over would occur, thus preventing false host protein identifications. The top 20 precursors were selected for MS2 data-dependent CID fragmentation. Spectra data was processed using Proteome Discoverer 1.4 with the SEQUEST search algorithm. The false discovery rate was calculated using PERCOLATOR and was set at <1% to score only high confidence peptide identifications.
Results:
Electron micrographs of the two-step purified SINV preparations showed extremely pure virus preparations, with no cellular debris or other cell-associated membrane-bound vesicles. Each purified viral preparation yielded unique host protein identifications with only a limited number host proteins being identified in the negative controls. Keratin was often identified in the negative control. This identification was ignored for functional interpretation and likely introduced during sample processing. The BHK21 hamster cells yielded over 50 unique protein IDs from the LC-MS/MS analysis. An additional 44 host proteins were identified from the HEK293 human background and 27 proteins from the C710 mosquito background. The proteins identified in each host background cover a wide range of molecular functions and protein classes. Proteins identified in both mammalian backgrounds have functions such as nucleic acid and protein binding, catalytic activity, regulator activity, receptor activity, and transporter activity. Collectively, they are members of classes such as chaperones, kinases, membrane traffic proteins, and signaling molecules. When comparing the BHK21 replicate virus preparations to each other, we identified four conserved proteins (sorting nexin-5, heat shock cognate 71, claudin, and junctional adhesion molecule), each with multiple peptides identified. In addition to comparing preparations within hosts, we also compared the host proteins identified between species, hamster versus human. Using this strategy we identified 6 protein signatures (sorting nexin-5, junctional adhesion molecule, cellular nucleic acid-binding protein, polyubiquitin, heat shock protein 70/71, and RNA binding protein 3) that were conserved between both host backgrounds.
Conclusions:
The development of antivirals, vaccines, and investigative tools for viruses represents a major gap in our ability to protect the public and the warfighter from emerging diseases. Basic understanding of virus-host biology remains central to addressing these gaps. Here, LC-MS/MS was utilized to determine if Sindbis virus specifically packages host proteins into its virus particles. Mass spectrometry analysis revealed that a variety of host proteins are being incorporated into SINV particles in each host background. Of note, when comparing the mammalian backgrounds, was the identification of a core group of conserved host proteins. One of these host proteins, sorting nexin 5 (SNX), has recently been elucidated as a restriction factor for Chlamydia infection.2,3 To counter SNX5’s restrictive effect, Chlamydia sequesters SNX5 in replicative bodies and prevents is action on membrane rearrangements required for new progeny. SINV also requires extensive membrane rearrangement for replication and assembly at the plasma membrane; therefore, SNX5 might be hijacked by SINV during particle assembly to induce events necessary for infectious particles to be formed. While SNX5 has been definitively implicated in infection, the other conserved proteins identified in this study have a less-clear potential role in SINV replication. Overall, this study has produced results that can significantly contribute to the understanding of alphavirus-host biology. The core host proteins identified in this study will serve as the molecular foundation for future research efforts centered on each protein’s functional role in the viral life cycle.
References & Acknowledgements:
References:
1.Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–62 (2009).
2.Aeberhard, L. et al. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components. PLOS Pathog. 11, e1004883 (2015).
3.Mirrashidi, K. M. et al. Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection. Cell Host Microbe 18, 109–21 (2015).
Acknowledgments:
This work was funded through the U.S. Army In-House Laboratory Independent Research (ILIR) program at the Edgewood Chemical Biological Center. The authors would like to thank Dr. Way Fountain, Dr. Nicole Rosenzweig, Ms. Stacey Broomall, and Ms. Rebecca Braun for continued scientific and administrative support.
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