Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.


Journal

Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555

Informations de publication

Date de publication:
14 12 2021
Historique:
received: 06 05 2021
accepted: 22 11 2021
entrez: 15 12 2021
pubmed: 16 12 2021
medline: 6 1 2022
Statut: epublish

Résumé

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

Identifiants

pubmed: 34907161
doi: 10.1038/s41467-021-27511-1
pii: 10.1038/s41467-021-27511-1
pmc: PMC8671429
doi:

Substances chimiques

Membrane Proteins 0
NS4B protein, Dengue virus 0
Phosphatidic Acids 0
Spike Glycoprotein, Coronavirus 0
Viral Nonstructural Proteins 0
Viral Proteins 0
spike protein, SARS-CoV-2 0
Acyltransferases EC 2.3.-
1-Acylglycerol-3-Phosphate O-Acyltransferase EC 2.3.1.51
AGPAT1 protein, human EC 2.3.1.51
2-acylglycerophosphate acyltransferase EC 2.3.1.52

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

7276

Subventions

Organisme : European Molecular Biology Organization (EMBO)
ID : ALTF 454-2020
Organisme : European Molecular Biology Organization (EMBO)
ID : ALTF 466-2016
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)
ID : Swiss National Science foundation (SNF Project Number 310030_173085
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)
ID : Swiss National Science foundation (SNF Project Number 310030_173085
Organisme : Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)
ID : grant number 031A602A (ERASysApp SysVirDrug)
Organisme : Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)
ID : grant number 031A602A (ERASysApp SysVirDrug)
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : Project Number 314905040 - TRR 209
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : Project Number 272983813 - TRR 179
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : Project Number 112927078 - TRR 83
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : Project Number 314905040 - TRR 209

Informations de copyright

© 2021. The Author(s).

Références

WHO Situation Reports. Coronavirus disease (COVID-19) Weekly Epidemiological Update and Weekly Operational Update.) (2021).
Spearman, C. W., Dusheiko, G. M., Hellard, M., Sonderup, M. & Hepatitis, C. Lancet 394, 1451–1466 (2019).
pubmed: 31631857 doi: 10.1016/S0140-6736(19)32320-7
Wolf, Y. I. et al. Origins and evolution of the Global RNA virome. mBio 9, e02329–18 (2018).
pubmed: 30482837 pmcid: 6282212 doi: 10.1128/mBio.02329-18
Neufeldt, C. J., Cortese, M., Acosta, E. G. & Bartenschlager, R. Rewiring cellular networks by members of the Flaviviridae family. Nat. Rev. Microbiol 16, 125–142 (2018).
pubmed: 29430005 pmcid: 7097628 doi: 10.1038/nrmicro.2017.170
V’Kovski, P., Kratzel, A., Steiner, S., Stalder, H. & Thiel, V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat. Rev. Microbiol 19, 155–170 (2021).
pubmed: 33116300 doi: 10.1038/s41579-020-00468-6
Lamb, C. A., Yoshimori, T. & Tooze, S. A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).
pubmed: 24201109 doi: 10.1038/nrm3696
Paul, D. & Bartenschlager, R. Flaviviridae replication organelles: oh, what a tangled web we weave. Annu. Rev. Virol. 2, 289–310 (2015).
pubmed: 26958917 doi: 10.1146/annurev-virology-100114-055007
Romero-Brey, I. et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog. 8, e1003056 (2012).
pubmed: 23236278 pmcid: 3516559 doi: 10.1371/journal.ppat.1003056
Cortese, M. et al. Integrative imaging reveals SARS-CoV-2-induced reshaping of subcellular morphologies. Cell Host Microbe 28, 853–866.e855 (2020).
pubmed: 33245857 pmcid: 7670925 doi: 10.1016/j.chom.2020.11.003
Snijder, E. J. et al. A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis. PLoS Biol. 18, e3000715 (2020).
pubmed: 32511245 pmcid: 7302735 doi: 10.1371/journal.pbio.3000715
Wolff, G., Melia, C. E., Snijder, E. J. & Barcena, M. Double-membrane vesicles as platforms for viral replication. Trends Microbiol. 28, 1022–1033 (2020).
pubmed: 32536523 pmcid: 7289118 doi: 10.1016/j.tim.2020.05.009
Oudshoorn, D. et al. Expression and cleavage of Middle East respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral RNA replication. mBio 8, e01658-17 (2017).
Paul, D., Hoppe, S., Saher, G., Krijnse-Locker, J. & Bartenschlager, R. Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J. Virol. 87, 10612–10627 (2013).
pubmed: 23885072 pmcid: 3807400 doi: 10.1128/JVI.01370-13
Yamashita, A. et al. Glycerophosphate/Acylglycerophosphate acyltransferases. Biology 3, 801–830 (2014).
pubmed: 25415055 pmcid: 4280512 doi: 10.3390/biology3040801
Takeuchi, K. & Reue, K. Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis. Am. J. Physiol. Endocrinol. Metab. 296, E1195–E1209 (2009).
pubmed: 19336658 pmcid: 2692402 doi: 10.1152/ajpendo.90958.2008
Kooijman, E. E., Chupin, V., de Kruijff, B. & Burger, K. N. Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid. Traffic 4, 162–174 (2003).
pubmed: 12656989 doi: 10.1034/j.1600-0854.2003.00086.x
Zegarlinska, J., Piascik, M., Sikorski, A. F. & Czogalla, A. Phosphatidic acid - a simple phospholipid with multiple faces. Acta Biochim Pol. 65, 163–171 (2018).
pubmed: 29913482 doi: 10.18388/abp.2018_2592
Tanguy, E., Wang, Q., Moine, H. & Vitale, N. Phosphatidic acid: from pleiotropic functions to neuronal pathology. Front. Cell Neurosci. 13, 2 (2019).
pubmed: 30728767 pmcid: 6351798 doi: 10.3389/fncel.2019.00002
Mizumura, K., Choi, A. M. & Ryter, S. W. Emerging role of selective autophagy in human diseases. Front. Pharm. 5, 244 (2014).
doi: 10.3389/fphar.2014.00244
Fernandez-Galilea, M., Tapia, P., Cautivo, K., Morselli, E. & Cortes, V. A. AGPAT2 deficiency impairs adipogenic differentiation in primary cultured preadipocytes in a non-autophagy or apoptosis dependent mechanism. Biochem. Biophys. Res. Commun. 467, 39–45 (2015).
pubmed: 26417690 doi: 10.1016/j.bbrc.2015.09.128
Cautivo, K. M. et al. AGPAT2 is essential for postnatal development and maintenance of white and brown adipose tissue. Mol. Metab. 5, 491–505 (2016).
pubmed: 27408775 pmcid: 4921804 doi: 10.1016/j.molmet.2016.05.004
Lee, J. Y. et al. Spatiotemporal coupling of the hepatitis C virus replication cycle by creating a lipid droplet- proximal membranous replication compartment. Cell Rep. 27, 3602–3617.e3605 (2019).
pubmed: 31216478 doi: 10.1016/j.celrep.2019.05.063
Khan, I. et al. Modulation of hepatitis C virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2. J. Virol. 88, 12276–12295 (2014).
pubmed: 25122779 pmcid: 4248901 doi: 10.1128/JVI.00970-14
Zhang, F. et al. Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells. Mol. Cell Biol. 34, 84–95 (2014).
pubmed: 24164897 pmcid: 3911278 doi: 10.1128/MCB.00987-13
Rizzo, M. A., Shome, K., Watkins, S. C. & Romero, G. The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras. J. Biol. Chem. 275, 23911–23918 (2000).
pubmed: 10801816 doi: 10.1074/jbc.M001553200
Prakash, P., Hancock, J. F. & Gorfe, A. A. Three distinct regions of cRaf kinase domain interact with membrane. Sci. Rep. 9, 2057 (2019).
pubmed: 30765804 pmcid: 6375958 doi: 10.1038/s41598-019-38770-w
Sakane, F., Hoshino, F. & Murakami, C. New era of diacylglycerol kinase, phosphatidic acid and phosphatidic acid-binding protein. Int. J. Mol. Sci. 21, 6794 (2020).
Yamano, K. et al. Endosomal Rab cycles regulate Parkin-mediated mitophagy. Elife 7, e31326 (2018).
Vargas, J. N. S. et al. Spatiotemporal control of ULK1 activation by NDP52 and TBK1 during selective autophagy. Mol. Cell 74, 347–362.e346 (2019).
pubmed: 30853401 pmcid: 6642318 doi: 10.1016/j.molcel.2019.02.010
Holland, P. et al. HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels. Nat. Commun. 7, 13889 (2016).
pubmed: 28004827 pmcid: 5412012 doi: 10.1038/ncomms13889
Knoops, K. et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 6, e226 (2008).
pubmed: 18798692 pmcid: 2535663 doi: 10.1371/journal.pbio.0060226
Wolff, G. et al. A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369, 1395–1398 (2020).
Welsch, S. et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5, 365–375 (2009).
pubmed: 19380115 pmcid: 7103389 doi: 10.1016/j.chom.2009.03.007
Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. & Chen, J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 1942–1945 (2001).
pubmed: 11729323 doi: 10.1126/science.1066015
Chu, J. Y. K. & Ou, J. J. Autophagy in HCV replication and protein trafficking. Int. J. Mol. Sci. 22, 1089 (2021).
Mori, H. et al. Induction of selective autophagy in cells replicating hepatitis C virus genome. J. Gen. Virol. 99, 1643–1657 (2018).
pubmed: 30311874 doi: 10.1099/jgv.0.001161
Shrivastava, S., Bhanja Chowdhury, J., Steele, R., Ray, R. & Ray, R. B. Hepatitis C virus upregulates Beclin1 for induction of autophagy and activates mTOR signaling. J. Virol. 86, 8705–8712 (2012).
pubmed: 22674982 pmcid: 3421755 doi: 10.1128/JVI.00616-12
Huang, H. et al. Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy. Autophagy 9, 175–195 (2013).
pubmed: 23169238 pmcid: 3552882 doi: 10.4161/auto.22791
Pagliuso, A. et al. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase delta. Nat. Commun. 7, 12148 (2016).
pubmed: 27401954 pmcid: 4945875 doi: 10.1038/ncomms12148
Jang, J.-H., Lee, C. S., Hwang, D. & Ryu, S. H. Understanding of the roles of phospholipase D and phosphatidic acid through their binding partners. Prog. Lipid Res. 51, 71–81 (2012).
Wang, H. & Tai, A. W. Nir2 is an effector of VAPs necessary for efficient hepatitis C virus replication and phosphatidylinositol 4-phosphate enrichment at the viral replication organelle. J. Virol. 93, e00742-19 (2019).
Judith, D. et al. ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIbeta. J. Cell Biol. 218, 1634–1652 (2019).
pubmed: 30917996 pmcid: 6504893 doi: 10.1083/jcb.201901115
Muller, C. et al. Inhibition of cytosolic phospholipase A2alpha impairs an early step of coronavirus replication in cell culture. J. Virol. 92, e01463-17 (2018).
Neufeldt, C. J. et al. ER-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly. Nat. Microbiol. 4, 2416–2429 (2019).
Stoeck, I. K. et al. Hepatitis C virus replication depends on endosomal cholesterol homeostasis. J. Virol. 92, e01196-17 (2018).
Fischl, W. & Bartenschlager, R. High-throughput screening using dengue virus reporter genomes. Methods Mol. Biol. 1030, 205–219 (2013).
pubmed: 23821271 doi: 10.1007/978-1-62703-484-5_17
Münster, M. et al. A reverse genetics system for zika virus based on a simple molecular cloning strategy. Viruses 10, 368 (2018).
pmcid: 6071187 doi: 10.3390/v10070368
Klein, S. et al. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nat. Commun. 11, 5885 (2020).
pubmed: 33208793 pmcid: 7676268 doi: 10.1038/s41467-020-19619-7
Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).
pubmed: 19377485 doi: 10.1038/nmeth.1322
Olsen, J. V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell Proteom. 4, 2010–2021 (2005).
doi: 10.1074/mcp.T500030-MCP200
Teo, G. et al. SAINTexpress: improvements and additional features in significance analysis of INTeractome software. J. Proteom. 100, 37–43 (2014).
doi: 10.1016/j.jprot.2013.10.023
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
pubmed: 25605792 pmcid: 4402510 doi: 10.1093/nar/gkv007
Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
pubmed: 14597658 pmcid: 403769 doi: 10.1101/gr.1239303
Ozbalci, C., Sachsenheimer, T. & Brugger, B. Quantitative analysis of cellular lipids by nano-electrospray ionization mass spectrometry. Methods Mol. Biol. 1033, 3–20 (2013).
pubmed: 23996167 doi: 10.1007/978-1-62703-487-6_1
Poenisch, M. et al. Identification of HNRNPK as regulator of hepatitis C virus particle production. PLoS Pathog. 11, e1004573 (2015).
pubmed: 25569684 pmcid: 4287573 doi: 10.1371/journal.ppat.1004573
Rieber, N., Knapp, B., Eils, R. & Kaderali, L. RNAither, an automated pipeline for the statistical analysis of high-throughput RNAi screens. Bioinformatics 25, 678–679 (2009).
pubmed: 19168909 doi: 10.1093/bioinformatics/btp014
Tabata, K. et al. Unique requirement for ESCRT factors in flavivirus particle formation on the endoplasmic reticulum. Cell Rep. 16, 2339–2347 (2016).
pubmed: 27545892 doi: 10.1016/j.celrep.2016.07.068
Prasad, V., Suomalainen, M., Hemmi, S. & Greber, U. F. Cell cycle-dependent kinase Cdk9 is a postexposure drug target against human adenoviruses. ACS Infect. Dis. 3, 398–405 (2017).
pubmed: 28434229 doi: 10.1021/acsinfecdis.7b00009
Neufeldt, C. J. et al. SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB. Preprint at https://search.bvsalud.org/global-literature-on-novel-coronavirus-2019-ncov/resource/en/ppbiorxiv-212639 (2020).
Schaller, T. et al. Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J. Virol. 81, 4591–4603 (2007).
pubmed: 17301154 pmcid: 1900174 doi: 10.1128/JVI.02144-06
Kano, F. et al. Hydrogen peroxide depletes phosphatidylinositol-3-phosphate from endosomes in a p38 MAPK-dependent manner and perturbs endocytosis. Biochim. Biophys. Acta 1813, 784–801 (2011).
pubmed: 21277337 doi: 10.1016/j.bbamcr.2011.01.023
Zettl, F. et al. Rapid quantification of SARS-CoV-2-neutralizing antibodies using propagation-defective vesicular stomatitis virus pseudotypes. Vaccines 8, 386 (2020).
Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).
pubmed: 30395289 doi: 10.1093/nar/gky1106

Auteurs

Keisuke Tabata (K)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.
Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

Vibhu Prasad (V)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

David Paul (D)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Ji-Young Lee (JY)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Minh-Tu Pham (MT)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Woan-Ing Twu (WI)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Christopher J Neufeldt (CJ)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Mirko Cortese (M)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Berati Cerikan (B)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Yannick Stahl (Y)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Sebastian Joecks (S)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
LI-COR Biosciences GmbH, Siemensstrasse 25A, Bad Homburg, Germany.

Cong Si Tran (CS)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Christian Lüchtenborg (C)

Biochemistry Center Heidelberg, Heidelberg University, Heidelberg, Germany.

Philip V'kovski (P)

Institute of Virology and Immunology IVI, Bern, Switzerland.
Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Katrin Hörmann (K)

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

André C Müller (AC)

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Carolin Zitzmann (C)

Institute of Bioinformatics and Center for Functional Genomics of Microbes, University Medicine Greifswald, Greifswald, Germany.
Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, NM, USA.

Uta Haselmann (U)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Jürgen Beneke (J)

BioQuant, Heidelberg University, Heidelberg, Germany.

Lars Kaderali (L)

Institute of Bioinformatics and Center for Functional Genomics of Microbes, University Medicine Greifswald, Greifswald, Germany.

Holger Erfle (H)

BioQuant, Heidelberg University, Heidelberg, Germany.

Volker Thiel (V)

Institute of Virology and Immunology IVI, Bern, Switzerland.
Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Volker Lohmann (V)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.

Giulio Superti-Furga (G)

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Britta Brügger (B)

Biochemistry Center Heidelberg, Heidelberg University, Heidelberg, Germany.

Ralf Bartenschlager (R)

Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany. ralf.bartenschlager@med.uni-heidelberg.de.
Division Virus-Associated Carcinogenesis, German Cancer Research Center, Heidelberg, Germany. ralf.bartenschlager@med.uni-heidelberg.de.
German Center for Infection Research, Heidelberg Partner Site, Heidelberg, Germany. ralf.bartenschlager@med.uni-heidelberg.de.

Articles similaires

[Redispensing of expensive oral anticancer medicines: a practical application].

Lisanne N van Merendonk, Kübra Akgöl, Bastiaan Nuijen
1.00
Humans Antineoplastic Agents Administration, Oral Drug Costs Counterfeit Drugs

Smoking Cessation and Incident Cardiovascular Disease.

Jun Hwan Cho, Seung Yong Shin, Hoseob Kim et al.
1.00
Humans Male Smoking Cessation Cardiovascular Diseases Female
Humans United States Aged Cross-Sectional Studies Medicare Part C
1.00
Humans Yoga Low Back Pain Female Male

Classifications MeSH