{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:29:21Z","timestamp":1785414561311,"version":"3.56.0"},"reference-count":46,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T00:00:00Z","timestamp":1784937600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"publisher","award":["R01 AI189249"],"award-info":[{"award-number":["R01 AI189249"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R01 AI189249"],"award-info":[{"award-number":["R01 AI189249"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"B-HIVE","doi-asserted-by":"publisher","award":["U54 AI170855"],"award-info":[{"award-number":["U54 AI170855"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The timing of HIV-1 protease (PR) activation and the determinants of orderly Gag and Gag-Pol polyprotein cleavage, leading to structural maturation of virions, are not well-understood. Here, we employed total internal reflection microscopy to visualize PR activation and cleavage of a FRET-based fluorescent substrate in single HIV-1 particles assembled on the cell\u2019s ventral membrane. PR activity manifested as a reduction in FRET signal in a relatively small fraction of nascent viral particles. By contrast, most virions released from cells contained a processed FRET substrate, suggesting that PR activation may be delayed or suppressed in most virions assembled at the ventral membrane. A combination of single-particle tracking and FRET measurements detected PR activity, on average, within ~20 min after the onset of particle assembly, while cleavage of a FRET substrate in single virions was completed within a few minutes. Importantly, substrates containing distinct PR cleavage sequences derived from Gag and Gag-Pol polyproteins were processed with different rates and efficiency. Our findings validate the use of surrogate FRET substrates to assess the timing of PR activation in single virions and elucidate the determinants of the tightly orchestrated order of Gag and Gag-Pol cleavage required for the formation of infectious virions.<\/jats:p>","DOI":"10.3390\/v18080819","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T16:11:56Z","timestamp":1785168716000},"page":"819","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Real-Time Imaging of HIV-1 Protease Activation and Substrate Cleavage in Single Virions Assembling on the Plasma Membrane"],"prefix":"10.3390","volume":"18","author":[{"given":"Yinglin","family":"Li","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8539-0295","authenticated-orcid":false,"given":"Satya P.","family":"Singh","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biophysics and Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariana","family":"Marin","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"},{"name":"Children\u2019s Healthcare of Atlanta, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alec L.","family":"Zhan","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashwanth C.","family":"Francis","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biophysics and Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5385-3013","authenticated-orcid":false,"given":"Gregory B.","family":"Melikyan","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"},{"name":"Children\u2019s Healthcare of Atlanta, Atlanta, GA 30322, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.sbi.2008.02.001","article-title":"The structural biology of HIV assembly","volume":"18","author":"Yeager","year":"2008","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1016\/j.jmb.2011.04.021","article-title":"The molecular architecture of HIV","volume":"410","author":"Briggs","year":"2011","journal-title":"J. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1038\/331280a0","article-title":"Characterization of ribosomal frameshifting in HIV-1 gag-pol expression","volume":"331","author":"Jacks","year":"1988","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"73","DOI":"10.2174\/1570162052772942","article-title":"The packaging and maturation of the HIV-1 Pol proteins","volume":"3","author":"Hill","year":"2005","journal-title":"Curr. HIV Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"40867","DOI":"10.1074\/jbc.R112.399444","article-title":"The choreography of HIV-1 proteolytic processing and virion assembly","volume":"287","author":"Lee","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kleinpeter, A.B., and Freed, E.O. (2020). HIV-1 Maturation: Lessons Learned from Inhibitors. Viruses, 12.","DOI":"10.3390\/v12090940"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"a006924","DOI":"10.1101\/cshperspect.a006924","article-title":"HIV-1 Assembly, Budding, and Maturation","volume":"2","author":"Sundquist","year":"2012","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1089\/aid.1989.5.577","article-title":"Cleavage of HIV-1 gag polyprotein synthesized in vitro: Sequential cleavage by the viral protease","volume":"5","author":"Manfredi","year":"1989","journal-title":"AIDS Res. Hum. Retroviruses"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Konnyu, B., Sadiq, S.K., Turanyi, T., Hirmondo, R., Muller, B., Krausslich, H.G., Coveney, P.V., and Muller, V. (2013). Gag-Pol processing during HIV-1 virion maturation: A systems biology approach. PLoS Comput Biol., 9.","DOI":"10.1371\/journal.pcbi.1003103"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Tien, C., Huang, L., Watanabe, S.M., Speidel, J.T., Carter, C.A., and Chen, C. (2018). Context-dependent autoprocessing of human immunodeficiency virus type 1 protease precursors. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0191372"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"13279","DOI":"10.1074\/jbc.M112.339374","article-title":"Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease","volume":"287","author":"Lee","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1186\/1742-4690-2-66","article-title":"Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates","volume":"2","author":"Pettit","year":"2005","journal-title":"Retrovirology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"eabe1707","DOI":"10.1126\/science.abe1707","article-title":"CARD8 is an inflammasome sensor for HIV-1 protease activity","volume":"371","author":"Wang","year":"2021","journal-title":"Science"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"198258","DOI":"10.1016\/j.virusres.2020.198258","article-title":"Conditional activation of an HIV-1 protease attenuated mutant by a leucine zipper dimerization motif","volume":"295","author":"Yu","year":"2021","journal-title":"Virus Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Figueiredo, A., Moore, K.L., Mak, J., Sluis-Cremer, N., de Bethune, M.P., and Tachedjian, G. (2006). Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol. PLoS Pathog., 2.","DOI":"10.1371\/journal.ppat.0020119"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Tabler, C.O., Wegman, S.J., Alhusaini, N., Lee, N.F., and Tilton, J.C. (2024). Premature Activation of the HIV-1 Protease Is Influenced by Polymorphisms in the Hinge Region. Viruses, 16.","DOI":"10.3390\/v16060849"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"13722","DOI":"10.1128\/JVI.02271-14","article-title":"Induced maturation of human immunodeficiency virus","volume":"88","author":"Mattei","year":"2014","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6461","DOI":"10.1038\/ncomms7461","article-title":"Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor","volume":"6","author":"Schimer","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.4155\/fmc.13.89","article-title":"Understanding HIV-1 protease autoprocessing for novel therapeutic development","volume":"5","author":"Huang","year":"2013","journal-title":"Future Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4477","DOI":"10.1074\/jbc.271.8.4477","article-title":"A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation","volume":"271","author":"Wondrak","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1021\/bi00171a027","article-title":"Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1","volume":"33","author":"Co","year":"1994","journal-title":"Biochemistry"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"7970","DOI":"10.1073\/pnas.91.17.7970","article-title":"Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein","volume":"91","author":"Louis","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1006\/viro.1993.1174","article-title":"Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles","volume":"193","author":"Karacostas","year":"1993","journal-title":"Virology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1834","DOI":"10.1128\/JVI.75.4.1834-1841.2001","article-title":"Maintenance of the Gag\/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity","volume":"75","author":"Crowe","year":"2001","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1186\/1742-4690-7-89","article-title":"Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease","volume":"7","author":"Jochmans","year":"2010","journal-title":"Retrovirology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/j.bpj.2020.06.023","article-title":"Dynamics of the HIV Gag Lattice Detected by Localization Correlation Analysis and Time-Lapse iPALM","volume":"119","author":"Saha","year":"2020","journal-title":"Biophys. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e0219821","DOI":"10.1128\/jvi.02198-21","article-title":"The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release","volume":"96","author":"Tabler","year":"2022","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6782","DOI":"10.1128\/jvi.68.10.6782-6786.1994","article-title":"The activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency","volume":"68","author":"Kaplan","year":"1994","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Qian, C., Flemming, A., Muller, B., and Lamb, D.C. (2022). Dynamics of HIV-1 Gag Processing as Revealed by Fluorescence Lifetime Imaging Microscopy and Single Virus Tracking. Viruses, 14.","DOI":"10.3390\/v14020340"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Bendjennat, M., and Saffarian, S. (2016). The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005657"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1016\/j.chom.2011.10.015","article-title":"Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion","volume":"10","author":"Dale","year":"2011","journal-title":"Cell Host Microbe"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Bendjennat, M., and Saffarian, S. (2019). Fluorescent Protein Inserts in between NC and SP2 are Tolerated for Assembly, Release and Maturation of HIV with Limited Infectivity. Viruses, 11.","DOI":"10.3390\/v11110973"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"21637","DOI":"10.1073\/pnas.2008156117","article-title":"Rapid formation of human immunodeficiency virus-like particles","volume":"117","author":"Bednarska","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.virol.2017.02.016","article-title":"High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry","volume":"505","author":"Bonar","year":"2017","journal-title":"Virology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"18101","DOI":"10.1038\/s41598-020-75118-1","article-title":"Nanoscale flow cytometry reveals interpatient variability in HIV protease activity that correlates with viral infectivity and identifies drug-resistant viruses","volume":"10","author":"Bonar","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"20196","DOI":"10.1074\/jbc.M117.818088","article-title":"An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions","volume":"292","author":"Sood","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"11736","DOI":"10.1038\/srep11736","article-title":"Identification of BST-2\/tetherin-induced hepatitis B virus restriction and hepatocyte-specific BST-2 inactivation","volume":"5","author":"Lv","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Mitchell, R.S., Katsura, C., Skasko, M.A., Fitzpatrick, K., Lau, D., Ruiz, A., Stephens, E.B., Margottin-Goguet, F., Benarous, R., and Guatelli, J.C. (2009). Vpu antagonizes BST-2-mediated restriction of HIV-1 release via \u03b2-TrCP and endo-lysosomal trafficking. PLoS Pathog., 5.","DOI":"10.1371\/journal.ppat.1000450"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1016\/j.cell.2009.08.039","article-title":"Tetherin inhibits HIV-1 release by directly tethering virions to cells","volume":"139","author":"Zang","year":"2009","journal-title":"Cell"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e34772","DOI":"10.7554\/eLife.34772","article-title":"Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis","volume":"7","author":"Marquez","year":"2018","journal-title":"eLife"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2928","DOI":"10.1021\/acsnano.3c07678","article-title":"HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice","volume":"18","author":"Gifford","year":"2024","journal-title":"ACS Nano"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e2019467118","DOI":"10.1073\/pnas.2019467118","article-title":"HIV-1 cores retain their integrity until minutes before uncoating in the nucleus","volume":"118","author":"Li","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3256","DOI":"10.1016\/j.bpj.2025.08.028","article-title":"3D localization of retrovirus assembly in the presence of structured background with deep learning","volume":"124","author":"Kohler","year":"2025","journal-title":"Biophys. J."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ivanchenko, S., Godinez, W.J., Lampe, M., Krausslich, H.G., Eils, R., Rohr, K., Brauchle, C., Muller, B., and Lamb, D.C. (2009). Dynamics of HIV-1 assembly and release. PLoS Pathog., 5.","DOI":"10.1371\/journal.ppat.1000652"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1038\/nature06998","article-title":"Imaging the biogenesis of individual HIV-1 virions in live cells","volume":"454","author":"Jouvenet","year":"2008","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"8036","DOI":"10.1016\/j.vaccine.2007.09.016","article-title":"Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines","volume":"25","author":"Hammonds","year":"2007","journal-title":"Vaccine"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/819\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T16:12:56Z","timestamp":1785168776000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/819"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,25]]},"references-count":46,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["v18080819"],"URL":"https:\/\/doi.org\/10.3390\/v18080819","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,25]]}}}