The matrix (MA) domain of the Mason-Pfizer monkey virus (M-PMV) Gag polyprotein plays a central role in retroviral assembly and trafficking, coordinating membrane association and proteolytic maturation. Unlike HIV-1, M-PMV assembles immature particles in the cytoplasm prior to plasma membrane targeting, but the molecular mechanisms governing this process remain poorly understood. Here, we identify calmodulin (CaM) as a calcium-dependent modulator of MA structural dynamics. Using a combination of biophysical and biochemical methods, we demonstrate that CaM directly interacts with myristoylated MA, promoting its oligomerization and enhancing its cleavage by the viral protease. In-depth characterization of MA-CaM complex by protein cross-linking mass spectrometry, hydrogen/deuterium exchange mass spectrometry and NMR spectroscopy reveals that the N-terminal parts of both proteins are in close proximity within the complex and that CaM binding induces increased conformational flexibility of key regions within MA, including the basic patch and C-terminal cleavage site. These dynamic changes suggest an allosteric mechanism by which CaM regulates MA function, potentially facilitating the temporal coordination of membrane targeting, the myristoyl switch and proteolytic processing. Our findings broaden the understanding of CaM as a regulatory factor in retroviral assembly and underscore the importance of conformational plasticity in viral maturation.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Czech Republic; Institute of Organic Chemistry and Biochemistry The Czech Academy of Science Prague Czech Republic

Institute of Microbiology The Czech Academy of Sciences Prague Czech Republic

Institute of Organic Chemistry and Biochemistry The Czech Academy of Science Prague Czech Republic

Institute of Organic Chemistry and Biochemistry The Czech Academy of Science Prague Czech Republic; Department of Analytical Chemistry Faculty of Science Charles University Prague Czech Republic

Zobrazit více v PubMed

Qu Y., Sun Y., Yang Z., Ding C. Calcium ions signaling: targets for attack and utilization by viruses. Front. Microbiol. 2022;13 PubMed PMC

Panda S., Behera S., Alam M.F., Syed G.H. Endoplasmic reticulum & mitochondrial calcium homeostasis: the interplay with viruses. Mitochondrion. 2021;58:227–242. PubMed PMC

Saimi Y., Kung C. Calmodulin as an ion channel subunit. Annu. Rev. Physiol. 2002;64:289–311. PubMed

Liu J.O. Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation. Immunol. Rev. 2009;228:184–198. PubMed PMC

Ross D.H., Cardenas H.L. Calmodulin stimulation of Ca2+-dependent ATP hydrolysis and ATP-dependent Ca2+ transport in synaptic membranes. J. Neurochem. 1983;41:161–171. PubMed

Virdi A.S., Singh S., Singh P. Abiotic stress responses in plants: roles of calmodulin-regulated proteins. Front Plant Sci. 2015;6:809. PubMed PMC

Pardo J.M., Reddy M.P., Yang S., Maggio A., Huh G.H., Matsumoto T., et al. Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates salt adaptation in plants. Proc Natl Acad Sci U. S. A. 1998;95:9681–9686. PubMed PMC

Liu S., Zhao L.Y., Cheng M.Z., Sun J.F., Ji X.M., Ullah A., et al. Calmodulins and calmodulin-like proteins-mediated plant organellar calcium signaling networks under abiotic stress. Crop. J. 2024;12:1321–1332.

Yang C.F., Tsai W.C. Calmodulin: the switch button of calcium signaling. Tzu Chi Med. J. 2022;34:15–22. PubMed PMC

Bhowmick R., Banik G., Chanda S., Chattopadhyay S., Chawla-Sarkar M. Rotavirus infection induces G1 to S phase transition in MA104 cells via Ca(+)(2)/Calmodulin pathway. Virology. 2014;454-455:270–279. PubMed PMC

Han Z., Harty R.N. Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway. Virus Genes. 2007;35:511–520. PubMed

Li F., Zhao N., Li Z., Xu X., Wang Y., Yang X., et al. A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana. PLoS Pathog. 2017;13 PubMed PMC

Tian W.J., Zhang X.Z., Wang J., Liu J.F., Li F.H., Wang X.J. Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response. Virol Sin. 2024;39:501–512. PubMed PMC

Mosialos G., Hanissian S.H., Jawahar S., Vara L., Kieff E., Chatila T.A. A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1. J Virol. 1994;68:1697–1705. PubMed PMC

Haolong C., Du N., Hongchao T., Yang Y., Wei Z., Hua Z., et al. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells. PLoS One. 2013;8 PubMed PMC

Chattopadhyay S., Basak T., Nayak M.K., Bhardwaj G., Mukherjee A., Bhowmick R., et al. Identification of cellular calcium binding protein Calmodulin as a regulator of rotavirus A infection during comparative proteomic study. PLoS One. 2013;8 PubMed PMC

Lewis M.G., Chang J.Y., Olsen R.G., Fertel R.H. Identification of calmodulin activity in purified retroviruses. Biochem. Biophys. Res. Commun. 1986;141:1077–1083. PubMed

Srinivas S.K., Srinivas R.V., Anantharamaiah G.M., Compans R.W., Segrest J.P. Cytosolic domain of the human immunodeficiency virus envelope glycoproteins binds to calmodulin and inhibits calmodulin-regulated proteins. J. Biol. Chem. 1993;268:22895–22899. PubMed

Radding W., Pan Z.Q., Hunter E., Johnston P., Williams J.P., McDonald J.M. Expression of HIV-1 envelope glycoprotein alters cellular calmodulin. Biochem. Biophys. Res. Commun. 1996;218:192–197. PubMed

Yuan T., Tencza S., Mietzner T.A., Montelaro R.C., Vogel H.J. Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41. Biopolymers. 2001;58:50–62. PubMed

Matsubara M., Jing T., Kawamura K., Shimojo N., Titani K., Hashimoto K., et al. Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo. Protein Sci. 2005;14:494–503. PubMed PMC

Radding W., Williams J.P., McKenna M.A., Tummala R., Hunter E., Tytler E.M., et al. Calmodulin and HIV type 1: interactions with Gag and Gag products. AIDS Res Hum Retroviruses. 2000;16:1519–1525. PubMed

Tzou Y.M., Shin R., Krishna N.R. HIV-1 virus interactions with host proteins: interaction of the N-terminal domain of the HIV-1 capsid protein with Human Calmodulin. Nat. Prod. Commun. 2019;14 PubMed PMC

Chow J.Y., Jeffries C.M., Kwan A.H., Guss J.M., Trewhella J. Calmodulin disrupts the structure of the HIV-1 MA protein. J. Mol. Biol. 2010;400:702–714. PubMed PMC

Ghanam R.H., Fernandez T.F., Fledderman E.L., Saad J.S. Binding of calmodulin to the HIV-1 matrix protein triggers myristate exposure. J. Biol. Chem. 2010;285:41911–41920. PubMed PMC

Vlach J., Samal A.B., Saad J.S. Solution structure of Calmodulin bound to the binding domain of the HIV-1 matrix protein. J. Biol. Chem. 2014;289:8697–8705. PubMed PMC

Samal A.B., Ghanam R.H., Fernandez T.F., Monroe E.B., Saad J.S. NMR, biophysical, and biochemical studies reveal the minimal Calmodulin binding domain of the HIV-1 matrix protein. J. Biol. Chem. 2011;286:33533–33543. PubMed PMC

Taylor J.E.N., Chow Y.H., John J.M., Cy K.H., Ann D.P., Anthony H.A., et al. Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release. Biophysical. J. 2012;103:541–549. PubMed PMC

Ihling C.H., Piersimoni L., Kipping M., Sinz A. Cross-Linking/Mass spectrometry combined with ion mobility on a timsTOF pro instrument for structural proteomics. Anal. Chem. 2021;93:11442–11450. PubMed

Konermann L., Metwally H., Duez Q., Peters I. Charging and supercharging of proteins for mass spectrometry: recent insights into the mechanisms of electrospray ionization. Analyst. 2019;144:6157–6171. PubMed

Osterholz H., Stevens A., Abramsson M.L., Lama D., Brackmann K., Rising A., et al. Native mass spectrometry captures the conformational plasticity of proteins with low-complexity domains. JACS Au. 2025;5:281–290. PubMed PMC

Castoralova M., Sys J., Prchal J., Pavlu A., Prokopova L., Briki Z., et al. A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein. Elife. 2024;13 PubMed PMC

Doktorova M., Heberle F.A., Kingston R.L., Khelashvili G., Cuendet M.A., Wen Y., et al. Cholesterol promotes protein binding by affecting membrane electrostatics and solvation properties. Biophys. J. 2017;113:2004–2015. PubMed PMC

Kalkhof S., Sinz A. Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters. Anal. Bioanal. Chem. 2008;392:305–312. PubMed

Prchal J., Srb P., Hunter E., Ruml T., Hrabal R. The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding. J. Mol. Biol. 2012;423:427–438. PubMed PMC

Labat-de-Hoz L., Comas L., Rubio-Ramos A., Casares-Arias J., Fernandez-Martin L., Pantoja-Uceda D., et al. Structure and function of the N-terminal extension of the formin INF2. Cell. Mol. Life. Sci. 2022;79:571. PubMed PMC

Proksova P.G., Lipov J., Zelenka J., Hunter E., Langerova H., Rumlova M., et al. Mason-Pfizer monkey virus Envelope Glycoprotein cycling and its vesicular Co-Transport with immature particles. Viruses. 2018;10 PubMed PMC

Ono A., Ablan S.D., Lockett S.J., Nagashima K., Freed E.O. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc. Natl. Acad. Sci. U. S. A. 2004;101:14889–14894. PubMed PMC

Kroupa T., Langerova H., Dolezal M., Prchal J., Spiwok V., Hunter E., et al. Membrane interactions of the mason-pfizer monkey virus matrix protein and its budding deficient mutants. J. Mol. Biol. 2016;428:4708–4722. PubMed

Tang C., Loeliger E., Luncsford P., Kinde I., Beckett D., Summers M.F. Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc. Natl. Acad. Sci. U. S. A. 2004;101:517–522. PubMed PMC

Fledderman E.L., Fujii K., Ghanam R.H., Waki K., Prevelige P.E., Freed E.O., et al. Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH. Biochemistry. 2010;49:9551–9562. PubMed PMC

Alfadhli A., Barklis R.L., Barklis E. HIV-1 matrix organizes as a hexamer of trimers on membranes containing phosphatidylinositol-(4,5)-bisphosphate. Virology. 2009;387:466–472. PubMed PMC

Srb P., Vlach J., Prchal J., Grocky M., Ruml T., Lang J., et al. Oligomerization of a retroviral matrix protein is facilitated by backbone flexibility on nanosecond time scale. J. Phys. Chem. B. 2011;115:2634–2644. PubMed PMC

Vlach J., Srb P., Prchal J., Grocky M., Lang J., Ruml T., et al. Nonmyristoylated matrix protein from the Mason-Pfizer monkey virus forms oligomers. J. Mol. Biol. 2009;390:967–980. PubMed

Johnson C.K., Harms G.S. Tracking and localization of calmodulin in live cells. Biochim. Biophys. Acta. 2016;1863:2017–2026. PubMed

Kishor C., Spillings B.L., Luhur J., Lutomski C.A., Lin C.H., McKinstry W.J., et al. Calcium contributes to polarized targeting of HIV assembly machinery by regulating complex stability. JACS Au. 2022;2:522–530. PubMed PMC

Contreras X., Bennasser Y., Chazal N., Moreau M., Leclerc C., Tkaczuk J., et al. Human immunodeficiency virus type 1 Tat protein induces an intracellular calcium increase in human monocytes that requires DHP receptors: involvement in TNF-alpha production. Virology. 2005;332:316–328. PubMed

Martoglio B., Graf R., Dobberstein B. Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin. EMBO J. 1997;16:6636–6645. PubMed PMC

Dostalkova A., Krizova I., Junkova P., Rackova J., Kapisheva M., Novotny R., et al. Unveiling the DHX15-G-patch interplay in retroviral RNA packaging. Proc. Natl. Acad. Sci. U. S. A. 2024;121 PubMed PMC

Bellacosa A., Testa J.R., Staal S.P., Tsichlis P.N. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science. 1991;254:274–277. PubMed

Ahmed N.N., Franke T.F., Bellacosa A., Datta K., Gonzalez-Portal M.E., Taguchi T., et al. The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene. 1993;8:1957–1963. PubMed

Bartoli M., Monneron A., Ladant D. Interaction of calmodulin with striatin, a WD-repeat protein present in neuronal dendritic spines. J. Biol. Chem. 1998;273:22248–22253. PubMed

Arbuzova A., Murray D., McLaughlin S. MARCKS, membranes, and calmodulin: kinetics of their interaction. Biochim. Biophys. Acta. 1998;1376:369–379. PubMed

Junkova P., Prchal J., Spiwok V., Pleskot R., Kadlec J., Krasny L., et al. Molecular aspects of the interaction between Mason-Pfizer monkey virus matrix protein and artificial phospholipid membrane. Proteins. 2016;84:1717–1727. PubMed

Chin D., Means A.R. Calmodulin: a prototypical calcium sensor. Trends Cell Biol. 2000;10:322–328. PubMed

Finn B.E., Forsen S. The evolving model of calmodulin structure, function and activation. Structure. 1995;3:7–11. PubMed

Villalobo A., Ishida H., Vogel H.J., Berchtold M.W. Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins. Biochim. Biophys. Acta, Mol. Cell Res. 2018;1865:507–521. PubMed

Prchal J., Sys J., Junkova P., Lipov J., Ruml T. Interaction interface of mason-pfizer monkey virus matrix and envelope proteins. J. Virol. 2020;94 PubMed PMC

Prchal J., Junkova P., Strmiskova M., Lipov J., Hynek R., Ruml T., et al. Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements. Protein Expr. Purif. 2011;79:122–127. PubMed PMC

Fuzik T., Ulbrich P., Ruml T. Efficient mutagenesis independent of ligation (EMILI) J Microbiol Methods. 2014;106:67–71. PubMed

Rhyner J.A., Koller M., Durussel-Gerber I., Cox J.A., Strehler E.E. Characterization of the human calmodulin-like protein expressed in Escherichia coli. Biochemistry. 1992;31:12826–12832. PubMed

Zabransky A., Andreansky M., Hruskova-Heidingsfeldova O., Havlicek V., Hunter E., Ruml T., et al. Three active forms of aspartic proteinase from Mason-Pfizer monkey virus. Virology. 1998;245:250–256. PubMed

Iacobucci C., Gotze M., Ihling C.H., Piotrowski C., Arlt C., Schafer M., et al. A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions. Nat. Protoc. 2018;13:2864–2889. PubMed

Kukacka Z., Rosulek M., Jelinek J., Slavata L., Kavan D., Novak P. LinX: a software tool for uncommon cross-linking chemistry. J. Proteome Res. 2021;20:2021–2027. PubMed

Perez-Riverol Y., Bai J., Bandla C., Garcia-Seisdedos D., Hewapathirana S., Kamatchinathan S., et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50:D543–D552. PubMed PMC

Honorato R.V., Koukos P.I., Jimenez-Garcia B., Tsaregorodtsev A., Verlato M., Giachetti A., et al. Structural biology in the clouds: the WeNMR-EOSC ecosystem. Front. Mol. Biosci. 2021;8 PubMed PMC

Honorato R.V., Trellet M.E., Jimenez-Garcia B., Schaarschmidt J.J., Giulini M., Reys V., et al. The HADDOCK2.4 web server for integrative modeling of biomolecular complexes. Nat. Protoc. 2024;19:3219–3241. PubMed

Meng E.C., Goddard T.D., Pettersen E.F., Couch G.S., Pearson Z.J., Morris J.H., et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 2023;32 PubMed PMC

Combe C.W., Graham M., Kolbowski L., Fischer L., Rappsilber J. xiVIEW: visualisation of crosslinking mass spectrometry data. J. Mol. Biol. 2024;436 PubMed