Jaisri Lingappa's Lab

Current Research Scientists

Jonathan C. Reed

Jonathan C. Reed

Jonathan C. Reed is a Senior Research Scientist in the Lingappa Lab.  Jon received his BS from Oregon State University (OSU), where he majored in Life Sciences/Botany and studied suppressors of RNA silencing encoded by plant viruses, in the laboratory of Dr. Valerian Dolja.  At OSU, Jon received numerous awards, including two HHMI summer undergraduate research fellowships and the Outstanding Senior Award at Oregon State University.  In 2004 Jon entered the University of Washington (UW) Molecular and Cellular Biology PhD program, and in 2006 he received an NSF Graduate Research Fellowship to pursue his PhD studies on capsid assembly in the Lingappa lab.   In 2011 he obtained his PhD in Molecular and Cellular Biology and his MS in Epidemiology through a joint UW PhD/MS program.  Jon stayed on in the Lingappa lab as a postdoctoral fellow until 2013, when he joined Prosetta Biosciences and established the Prosetta satellite lab in Seattle.  While at Prosetta, Jon served as Biology Lead and directed two different groups – one screening for anti-viral compounds that act on the subcellular ribonucleoprotein assembly machines that Jon studied while he was in the Lingappa lab, and one screening for anti-parasitic compounds that act on similar ribonucleoprotein complexes in the malaria parasite.  In 2017, Jon returned to Jaisri Lingappa’s lab at UW as a Senior Research Scientist to continue his studies of antiretroviral compounds in collaboration with Prosetta and to study the basic science of retroviral assembly.

Research:

As a PhD student in the Lingappa lab, Jon made a fundamental discovery that reshaped our understanding of HIV-1 virus assembly.  He demonstrated that HIV-1 assembly intermediates (aka assembly machines), which form during assembly of the HIV-1 immature capsid, are composed of cellular proteins found in RNA granules.  RNA granules are important subcellular host ribonucleoprotein complexes involved in all aspects of RNA metabolism except translation, but had not been implicated previously in HIV-1 assembly.  Jon also examined the role of these host proteins in HIV-1 assembly using siRNA knockdown approaches.  In these studies, he demonstrated that DDX6, one of the host RNA granule proteins he identified in HIV-1 assembly intermediates, facilitates HIV-1 capsid assembly in cells.  He further found that WT DDX6 rescued the knockdown phenotype, while an enzymatically inactive DDX6 mutant did not, thereby demonstrating that DDX6 acts enzymatically to promote HIV-1 assembly.  Jon confirmed the physiological relevance of his findings with siRNA knockdowns in primary blood mononuclear cells (PBMC), showing that DDX6 facilitates assembly of infectious virus.  Jon’s seminal PhD studies, published in the Journal of Cell Biology in 2012, support of a model in which HIV-1 Gag co-opts host RNA granules to form assembly intermediates and uses the host proteins in these complexes to facilitate capsid assembly, thereby adding to growing evidence that HIV-1 capsid assembly in cells is host-catalyzed and energy-dependent.

As a postdoctoral fellow, Jon developed RT-qPCR techniques to allow us to ask whether HIV-1 assembly machines also facilitate packaging of the HIV-1 genome into newly assembled capsids.  These advances led to a paper, published in PLOS Pathogens in 2018, showing that the RNA-granule-derived HIV-1 assembly intermediates contain much of the cytoplasmic HIV-1 genomic RNA found in HIV-expressing cells.  Thus, by trafficking to these RNA granules, assembling HIV-1 Gag is able to access host facilitators of assembly (e.g. DDX6 and the host enzyme ABCE1) as well as the HIV-1 genomic RNA that must be encapsidated during assembly for the resulting virus to be infectious.

As a Senior Research Scientist in the Lingappa Lab, Jon demonstrated that the Gag protein of feline immunodeficiency virus (FIV), a non-primate lentivirus, forms RNA-granule-derived assembly intermediates containing ABCE1 and DDX6, analogous to what is observed for HIV-1.  These findings suggest that retroviral assembly machines are evolutionarily conserved.  This study, published in the Journal of Virology in 2018, also demonstrated that the RNA-granule-derived FIV assembly intermediates contain additional RNA granule proteins, such as DCP-2, but do not contain the abundant ribosomal protein S6.  Thus, these complexes resemble RNA granules but do not resemble ribosomes.  This paper also features imaging studies in which Jon used the proximity ligation assay to demonstrate colocalization of FIV Gag with RNA granule proteins.

Jon’s most recent project describes a novel small molecule (PAV117), discovered in collaboration with Prosetta Biosciences, that inhibits replication of HIV-1 in T cell lines and primary T cells with nanomolar potency, most likely acting on HIV-1 assembly intermediates (manuscript in preparation).

Jon’s contributions to the Lingappa Lab are myriad, and include writing manuscripts, editing grants, supervising junior employees, helping to plan long-term research goals, generating ideas for new projects, researching the literature, and developing new methods.  He has piloted, optimized, and established a vast number of diverse techniques in the Lingappa lab, including highly sensitive coimmunoprecipitation approaches, quantitative immunogold electron microscopic double labeling, quantitative proximity ligation assays for studying subcellular localization of small molecules, siRNA knockdown and rescue, codon optimization, reverse transcription with quantitative PCR, northern blotting, a wide variety of infectivity assays, other virologic assays, and assays for studying inhibition of viral replication by antiviral small molecules including in primary cells.

Publications:

  1. Michon, M, Müller-Schiffmann, A, Lingappa, AF, Yu, SF, Du, L, Deiter, F, Broce, S, Mallesh, S, Crabtree, J, Lingappa, UF, Macieik, A, Müller, L, Ostermann, PN, Andrée, M, Adams, O, Schaal, H, Hogan, RJ, Tripp, RA, Appaiah, U, Anand, SK, Campi, TW, Ford, MJ, Reed, JC, Lin, J, Akintunde, O, Copeland, K, Nichols, C, Petrouski, E, Moreira, AR, Jiang, IT, DeYarman, N, Brown, I, Lau, S, Segal, I, Goldsmith, D, Hong, S, Asundi, V, Briggs, EM, Phyo, NS, Froehlich, M, Onisko, B, Matlack, K, Dey, D, Lingappa, JR, Prasad, MD, Kitaygorodskyy, A, Solas, D, Boushey, H, Greenland, J, Pillai, S, Lo, MK, Montgomery, JM, Spiropoulou, CF, Korth, C, Selvarajah, S, Paulvannan, K, Lingappa, VR. A Pan-Respiratory Antiviral Chemotype Targeting a Host Multi-Protein Complex. bioRxiv. 2023; :. doi: 10.1101/2021.01.17.426875. PubMed PMID:34931190 PubMed Central PMC8687465.
  2. Lingappa, JR, Lingappa, VR, Reed, JC. Addressing Antiretroviral Drug Resistance with Host-Targeting Drugs-First Steps towards Developing a Host-Targeting HIV-1 Assembly Inhibitor. Viruses. 2021;13 (3):. doi: 10.3390/v13030451. PubMed PMID:33802145 PubMed Central PMC8001593.
  3. Reed, JC, Solas, D, Kitaygorodskyy, A, Freeman, B, Ressler, DTB, Phuong, DJ, Swain, JV, Matlack, K, Hurt, CR, Lingappa, VR, Lingappa, JR. Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly. J Virol. 2021;95 (3):. doi: 10.1128/JVI.00883-20. PubMed PMID:33148797 PubMed Central PMC7925099.
  4. Barajas, BC, Tanaka, M, Robinson, BA, Phuong, DJ, Chutiraka, K, Reed, JC, Lingappa, JR. Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging. PLoS Pathog. 2018;14 (4):e1006977. doi: 10.1371/journal.ppat.1006977. PubMed PMID:29664940 PubMed Central PMC5940231.
  5. Reed, JC, Westergreen, N, Barajas, BC, Ressler, DTB, Phuong, DJ, Swain, JV, Lingappa, VR, Lingappa, JR. Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus. J Virol. 2018;92 (9):. doi: 10.1128/JVI.01761-17. PubMed PMID:29467316 PubMed Central PMC5899207.
  6. Tanaka, M, Robinson, BA, Chutiraka, K, Geary, CD, Reed, JC, Lingappa, JR. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins. J Virol. 2016;90 (4):1944-63. doi: 10.1128/JVI.02698-15. PubMed PMID:26656702 PubMed Central PMC4734008.
  7. Lingappa, JR, Reed, JC, Tanaka, M, Chutiraka, K, Robinson, BA. How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle. Virus Res. 2014;193 :89-107. doi: 10.1016/j.virusres.2014.07.001. PubMed PMID:25066606 PubMed Central PMC4351045.
  8. Robinson, BA, Reed, JC, Geary, CD, Swain, JV, Lingappa, JR. A temporospatial map that defines specific steps at which critical surfaces in the Gag MA and CA domains act during immature HIV-1 capsid assembly in cells. J Virol. 2014;88 (10):5718-41. doi: 10.1128/JVI.03609-13. PubMed PMID:24623418 PubMed Central PMC4019110.
  9. Reed, JC, Molter, B, Geary, CD, McNevin, J, McElrath, J, Giri, S, Klein, KC, Lingappa, JR. HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly. J Cell Biol. 2012;198 (3):439-56. doi: 10.1083/jcb.201111012. PubMed PMID:22851315 PubMed Central PMC3413349.
  10. Klein, KC, Reed, JC, Tanaka, M, Nguyen, VT, Giri, S, Lingappa, JR. HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag. J Virol. 2011;85 (14):7419-35. doi: 10.1128/JVI.00288-11. PubMed PMID:21543480 PubMed Central PMC3126549.
  11. Klein, KC, Reed, JC, Lingappa, JR. Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag. AIDS Rev. 2007;9 (3):150-61. . PubMed PMID:17982940 .
  12. Dooher, JE, Schneider, BL, Reed, JC, Lingappa, JR. Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production. Traffic. 2007;8 (3):195-211. doi: 10.1111/j.1600-0854.2006.00524.x. PubMed PMID:17233757 PubMed Central PMC1865004.
  13. Chiba, M, Reed, JC, Prokhnevsky, AI, Chapman, EJ, Mawassi, M, Koonin, EV, Carrington, JC, Dolja, VV. Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology. 2006;346 (1):7-14. doi: 10.1016/j.virol.2005.09.068. PubMed PMID:16300814 .
  14. Tzanetakis, IE, Reed, J, Martin, RR. Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus. Arch Virol. 2005;150 (2):273-86. doi: 10.1007/s00705-004-0410-z. PubMed PMID:15503221 .
  15. Reed, JC, Kasschau, KD, Prokhnevsky, AI, Gopinath, K, Pogue, GP, Carrington, JC, Dolja, VV. Suppressor of RNA silencing encoded by Beet yellows virus. Virology. 2003;306 (2):203-9. doi: 10.1016/s0042-6822(02)00051-x. PubMed PMID:12642093 .

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