Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy*

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5–/–, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5–/–cells. In MHV-infected APG5–/–cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

[1]  Obi L. Griffith,et al.  The Genome Sequence of the SARS-Associated Coronavirus , 2003, Science.

[2]  Xiaotao Lu,et al.  Intracellular andin Vitro-Translated 27-kDa Proteins Contain the 3C-like Proteinase Activity of the Coronavirus MHV-A59 , 1996, Virology.

[3]  N. Mizushima,et al.  Formation of the ∼350-kDa Apg12-Apg5·Apg16 Multimeric Complex, Mediated by Apg16 Oligomerization, Is Essential for Autophagy in Yeast* , 2002, The Journal of Biological Chemistry.

[4]  A. Sims,et al.  Mouse Hepatitis Virus Replicase Proteins Associate with Two Distinct Populations of Intracellular Membranes , 2000, Journal of Virology.

[5]  J. A. Comer,et al.  A novel coronavirus associated with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[6]  K. Fujiwara,et al.  Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture , 2005, Archiv für die gesamte Virusforschung.

[7]  K. Bienz,et al.  RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles , 2002, Journal of Virology.

[8]  Naoaki Goto,et al.  マウス肝炎ウイルス, MHV-2, MHV-3株の持続感染DBT細胞の樹立 , 1984 .

[9]  M. Denison,et al.  Four Proteins Processed from the Replicase Gene Polyprotein of Mouse Hepatitis Virus Colocalize in the Cell Periphery and Adjacent to Sites of Virion Assembly , 2000, Journal of Virology.

[10]  J. Ziebuhr,et al.  Mutational analysis of the active centre of coronavirus 3C-like proteases. , 2002, The Journal of general virology.

[11]  Takeshi Tokuhisa,et al.  Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells , 2001, The Journal of cell biology.

[12]  K. Kirkegaard,et al.  Cellular origin and ultrastructure of membranes induced during poliovirus infection , 1996, Journal of virology.

[13]  Y. Guan,et al.  Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage , 2003, Journal of Molecular Biology.

[14]  D. Klionsky,et al.  Autophagy in the Eukaryotic Cell , 2002, Eukaryotic Cell.

[15]  T. Takizawa,et al.  Virus Clearance through Apoptosis-Dependent Phagocytosis of Influenza A Virus-Infected Cells by Macrophages , 2000, Journal of Virology.

[16]  Y. van der Meer,et al.  Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex , 1999, Journal of Virology.

[17]  D. Scheuner,et al.  Regulation of starvation- and virus-induced autophagy by the eIF2α kinase signaling pathway , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Kirkegaard,et al.  Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles , 2000, Journal of Virology.

[19]  Xiaotao Lu,et al.  Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59 , 1995, Journal of virology.

[20]  S. Baker,et al.  Determinants of the p28 Cleavage Site Recognized by the First Papain-like Cysteine Proteinase of Murine Coronavirus , 1994, Virology.

[21]  S. Kyuwa,et al.  Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus , 1996, Journal of virology.

[22]  A. Sims,et al.  Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro , 1998, Journal of Virology.

[23]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[24]  S. Emr,et al.  Autophagy as a regulated pathway of cellular degradation. , 2000, Science.

[25]  Christian Drosten,et al.  Identification of a novel coronavirus in patients with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[26]  Arthur S Slutsky,et al.  Identification of severe acute respiratory syndrome in Canada. , 2003, The New England journal of medicine.

[27]  J. Ziebuhr,et al.  Conservation of substrate specificities among coronavirus main proteases. , 2002, The Journal of general virology.

[28]  M. Lai,et al.  Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells , 1999, Journal of Virology.

[29]  S. Weiss,et al.  Localization of mouse hepatitis virus open reading frame 1A derived proteins. , 1998, Journal of Neurovirology.

[30]  James E. Goldman,et al.  Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein , 1998, Journal of Virology.

[31]  M. Denison,et al.  Mouse Hepatitis Virus Replicase Protein Complexes Are Translocated to Sites of M Protein Accumulation in the ERGIC at Late Times of Infection , 2001, Virology.

[32]  A. Sims,et al.  The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis , 1999, Journal of Virology.

[33]  M. Denison,et al.  Determinants of Mouse Hepatitis Virus 3C-like Proteinase Activity , 1997, Virology.

[34]  M. Denison,et al.  MHV-A59 gene 1 proteins are associated with two distinct membrane populations. , 2001, Advances in experimental medicine and biology.

[35]  S. Kyuwa.  Replication of murine coronaviruses in mouse embryonic stem cell lines in vitro. , 1997, Experimental animals.