Principal Member of the Technical Staff
Oscar Negrete is a virologist with a special interest in emerging viruses and a focus on molecular viral-host interactions. Current model viruses of interest include Nipah virus, Ebola virus, Rift Valley fever virus, arenaviruses, flaviviruses, and alphaviruses. His research uses a broad spectrum of cellular, molecular biology, functional genomics, and nanoscience techniques to dissect host pathways involved in virus replication and deliver anti-viral cargos using nanoparticles. Pseudotyped systems are used for studying highly pathogenic viruses under BSL-2 containment. As a visiting scientist at the Lawrence Livermore National Laboratory, he has access to animal models and BSL-3 select agent research facilities. The ultimate goal of his research is to assist in the formulation of new countermeasures and diagnostics for emerging infectious disease pathogens.
Bachelor’s Degree: Microbiology and Molecular Genetics, University of California-Los Angeles (1999)
Doctoral Degree: Microbiology, Immunology, and Molecular Genetics, University of California-Los Angeles (2007); Advisor: Dr. Benhur Lee
Postdoctoral Fellowships: Virology, Sandia National Laboratories (2013)
Nipah and Hendra Virus
As a graduate student at UCLA, Negrete studied the entry mechanisms of Nipah virus (NiV) and Hendra virus (HeV), two emerging zoonotic paramyoxviruses in the genus Henipavirus. NiV infection in humans is highly fatal and results primarily in severe encephalitis. In efforts to understand henipavirus pathology and zoonotic transmission, he sought to identify the host receptors. Using immunoadhesin pulldown assays, he first determined that ephrinB2, a ligand for the Eph receptor tyrosine kinases, is a functional receptor for NiV. Expression of ephrinB2 in non-permissive cells allowed for NiV fusion and entry, while soluble ephrinB2 inhibited NiV fusion and entry. EphrinB2 is an evolutionary conserved protein expressed in neurons, endothelial and smooth muscle cells, and its discovery as a receptor provided an explanation for NiV tropism and zoonosis. He then identified ephrinB3, a homolog of ephrinB2, as an alterative receptor for NiV. EphrinB3 bound to NiV-G with subnanomolar affinity, albeit at a lower efficiency than ephrinB2. EphrinB3 was sufficient to allow entry of live NiV. Furthermore, mutational analysis indicated two overlapping ephrinB2 and B3 residues as critical determinants of NiV binding and entry. Global expression analysis showed distinct ephrinB3 expression in the brain stem. Thus, ephrinB3-mediated viral entry and pathology may underlie the severe brain stem neuronal dysfunction seen in fatal Nipah encephalitis. Lastly, he compared the efficiencies of ephrin receptor usage between henipaviruses and found they had comparable ephrinB2 receptor usage. However, ephrinB3 usage by HeV was significantly reduced compared to NiV. One residue, in an ephrinB3-specific binding domain, was found to cause this reduction. The differential usage of ephrinB3 by variant HeV strains may contribute to the differential pathological profiles observed between henipaviruses. Without vaccines or effective therapeutics, henipaviruses continue to present a critical threat to global health and economies. The identification of the receptors and the development of systems used in their discovery have not only shed light on certain aspects of human pathology caused by these viruses, but have also provided new strategies for therapeutic interventions.
- Negrete OA, MC Wolf, HC Aguilar, S Enterlein, W Wang, E Muhlberger, SV Su, A Bertolotti-Ciarlet, R Flick, B Lee. (2006). Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathogens 2:e7.
- Negrete OA, D Chu, HC Aguilar, B Lee. (2007). Single amino acid changes in the Nipah and Hendra virus attachment glycoprotein distinguishes ephrinB2 from ephrinB3 usage. Journal of Virology 81:10804.
- Lee, B, OA Negrete. Henipavirus receptor and uses thereof. International Patent publication WO/2007/005244. Jan 11, 2007.
- Maar D, Harmon B, Chu D, Shultz B, Aguilar HC, Lee B, Negrete OA*. (2012) Cysteines in the stalk domain of the Nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation. Journal of Virology 86(12):6632-42. *Corresponding author
Virology and Rift Valley Fever Virus
As a postdoctoral appointee, Negrete was hired at Sandia National Laboraotories to establish virology research in a department whose interests were mainly engineering, microfluidics, and diagnostics. Early in his postdoc, Negrete was awarded an internal grant to conduct virology research as a principal investigator and led a team of postdocs and technicans on a project focused on functional genomics screening using RNA interference (RNAi). Genome-wide RNAi screening offers a systematic investigation of critical cellular hostpathogen interactions, which in turn can serve to inform host-direct countermeasure development. Using this method against Rift Valley fever virus (RVFV), a biodefense threat listed as a category A priority pathogen, he identified initial candidate genes that helped dissect RVFV entry. To determine the cellular entry mechanism of RVFV, he used small-molecule inhibitors and dominant negative protein expression, in addition to RNAi to inhibit the major mammalian cell endocytic pathways. He found that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis. These results were in contrast to the original assumptions in the field prior to this study that suggested RVFV entry was mediated by clathrin dependent endocytosis.
By instituting the procedures for RNAi screening using traditional methodology, Negrete then integrated with the engineering team at Sandia to develop a miniaturized microfludic RNAi screening platform. Negrete and his team developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry using polydimethylsiloxane (PDMS)-based platforms. Validation of the screening platform was examined using Vesicular stomatitis virus and RVFV. The ultimate vision for this project was to enable large scale screening in high containment biosafety laboratories with portable inexpensive microfluidic platforms.
Lastly, using bioinformatic analysis of our entire genome wide screening results against RVFV, they found that the Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, they found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was pre-activated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. They ended up proposing a model where bunyaviruses activated Wnt responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication.
- Schudel BR, Harmon B, Abhyankar V, Pruitt B, Negrete OA*, Singh AK*. (2013) Microfluidic platforms for RNA Interference screening of virus-host interactions. Lab on a chip. Feb 5;13(5):811-7 (Back cover) *Co-Corresponding author.
- Perroud T, Renzi R, Negrete O, Claudnic MR. A Microelectroporation device for genomic screening.U.S. Patent 8,828,736. September 9,2014.
- Harmon B, Bird SW, Schudel BR, Hatch AV, Rasley A, Negrete OA* (2016) A genome-wide RNA interference screen identifies a role for Wnt/beta-catenin signaling during Rift Valley fever virus infection. Journal of Virology. July27;90(16):7084-97. *Corresponding author
Viral Research Models
As a permanent staff member at Sandia National Labs, Negrete has expanded Sandia’s catalog of viral research models and established collaborations for high containment-select agent research and for developing novel diagnostics, countermeasures and in vivo delivery vehicles to detect and treat biodefense and emerging viral diseases. Negrete and his team have now published several peer review manuscripts using various viral model systems that include henipaviruses, buynaviruses, arenaviruses, flaviviruses, alphaviruses, and filoviruses. Through a DTRA funded effort, they established collaborations with Dr. Amy Rasley at the Lawrence Livermore National Laboratory (LLNL) and with Dr. Jeff Brinker, a Distinguished and Regent’s Professor at the University of New Mexico (UNM) who has made numerous pioneering contributions to processing, characterization, and understanding of porous and composite nanostructured materials. The DTRA project goals were to develop lipid-coated mesoporous silica nanoparticles (LC-MSNs) as antiviral delivery vehicles targeting the central nervous system aimed at inhibiting encephalitic alphavirus infections. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal platform for antiviral drug delivery. At LLNL, in vitro antiviral testing was performed at BSL-3 against Venezuelan equine encephalitis virus (VEEV) and in vivo testing was performed using a vaccine strain of VEEV under ABSL-2 containment. Antiviral-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV TC-83 infected mice as compared to PBS treated controls and the manuscript describing the results is currently under review.
Negrete has also recently established collaboration with Jennifer Doudna, a professor at the University of California, Berkeley (UCB), HHMI investigator, Director of the Innovative Genomics Institute, and co-inventor of CRISPR-Cas9 genome-editing technology. Together, they have a DARPA-sponsored effort to develop CRISPRbased countermeasures to inhibit viral infection of biodefense concern, develop anti-CRISPR proteins to regulate genome editing activity, and to refine LC-MSN technology for in vivo CRISPR and anti-CRISPR cargo delivery. They have recently published an article in eLife describing RNA-dependent RNA targeting by CRISPRCas9 enzymes for Staphylococcus aureus and Campylobacter jejuni. Using the rules defined in this study, they have enabled transcriptional control in mammalian cells using RNA targeting Cas9 systems.
- Harmon B, Kozina C, Maar D, Carpenter T, Branda C, Carson B#, Negrete OA*#. (2013) Identification of amino acids within the nucleoprotein of Tacaribe virus important for anti interferon activity. Journal of Biological Chemistry. March22;288(12):8702-11 *Corresponding author, # Contributed equally
- Ashely C, Brinker CJ, Carnes EC, Fekrazad MH, Felton LA, Negrete O, Padilla DP, Wilkinson BS, Wilkinson DC, Willman CL. Porous nanoparticle-supported lipid bilayers (protocells) for targeted delivery including transdermal delivery of cargo and methods thereof. International Patent publication WO 2013056132. April 18, 2013.
- Meagher RJ, Negrete OA, Van Rompay KK. (2016) Engineering paper-based sensors for Zika virus.Trends in Molecular Medicine. July;22(7):529-30
- Malmlov A, Seetahal J, Carrington C, Ramkisson V, Foster J, Miazgowicz KL, Quackenbush S, Rovnak J, Negrete OA, Munster V, Schountz T. (2017) Serological evidence of arenavirus circulation among fruit bats in Trinidad. PLoS ONE 12(9). e0185308
- LaBauve A, Rinker T, Noureddine A, Serda R, Sherman M, Rasley A, Brinker CJ, Negrete OA. (2018) Lipid-coated mesoporous silica nanoparticles for the delivery of the ML336 antiviral to inhibit encephalitic alphavirus infection. Scientific Reports.
Relevant Prior Positions
- Graduate Student Researcher, University of California Los Angeles (2003-2007)
- Postdoctoral Appointee, Sandia National Laboratories (2008-2013)
- Senior Member of the Technical Staff, Sandia National Laboratories (2013-2018)
- Visiting Scientist, Livermore National Laboratory (2013-Present)
- Principal Member of Technical Staff, Sandia National Laboratories (2018-Present)
Honors and Memberships
- NIH Training Grant in Cellular Molecular Biology (2003-2006)
- UCLA Dissertation Year Fellowship (2006-2007) NIH Training Grant in Virology and Gene Therapy (2007-2008)
- Gordon Conference Carl Storm Underrepresented Minority Fellowship (2009)
- Sandia Post-Doctoral Technical Showcase Award (2010) Sandia Employee Recognition Award (2013)
- Hispanic Engineer National Achievement Award Conference Nomination (2015, 2016)
- FLC Outstanding Technology Development Award – SmartLamp Team (2017)
- Full Member, American Society for Microbiology (2013-Present)
- Full Member, American Society for Virology (2013-Present)