{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T02:16:58Z","timestamp":1781576218687,"version":"3.54.5"},"reference-count":57,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,9,13]],"date-time":"2020-09-13T00:00:00Z","timestamp":1599955200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R15AI123993-01A1"],"award-info":[{"award-number":["R15AI123993-01A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["HDTRA11810040 (A.N.)"],"award-info":[{"award-number":["HDTRA11810040 (A.N.)"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["HDTRA1-15-01-0047 (W.B.K.)"],"award-info":[{"award-number":["HDTRA1-15-01-0047 (W.B.K.)"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-\u03baB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKK\u03b2 inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKK\u03b2 and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKK\u03b2 kinase activity can directly phosphorylate VEEV nsP3 at sites 204\/5, 142, and 134\/5. Alanine substitution mutations at sites 204\/5, 142, or 134\/5 reduced VEEV replication by &gt;30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204\/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204\/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134\/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKK\u03b2 can phosphorylate VEEV nsP3 at sites 204\/5, 142, and 134\/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204\/5, but may be important for infectious particle production at site 134\/5.<\/jats:p>","DOI":"10.3390\/v12091021","type":"journal-article","created":{"date-parts":[[2020,9,13]],"date-time":"2020-09-13T21:11:32Z","timestamp":1600031492000},"page":"1021","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKK\u03b2 Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2169-756X","authenticated-orcid":false,"given":"Allison","family":"Bakovic","sequence":"first","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nishank","family":"Bhalla","sequence":"additional","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Kortchak","sequence":"additional","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengqun","family":"Sun","sequence":"additional","affiliation":[{"name":"Center for Vaccine Research, Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weidong","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Systems Biology, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aslaa","family":"Ahmed","sequence":"additional","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6999-7564","authenticated-orcid":false,"given":"Kenneth","family":"Risner","sequence":"additional","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William B.","family":"Klimstra","sequence":"additional","affiliation":[{"name":"Center for Vaccine Research, Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aarthi","family":"Narayanan","sequence":"additional","affiliation":[{"name":"National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,9,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1128\/mr.58.3.491-562.1994","article-title":"The alphaviruses: Gene expression, replication, and evolution","volume":"58","author":"Strauss","year":"1994","journal-title":"Microbiol. 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