Initiation of hepatitis C virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein.
Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified beta- and alpha-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.
Roohvand
Farzin
F
Unité des Hépacivirus et Immunité Innée and Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France.
Maillard
Patrick
P
Unité des Hépacivirus et Immunité Innée and Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France.
Lavergne
Jean-Pierre
JP
Institut de Biologie et Chimie de Protéines (IBCP-UMR 5086), CNRS, Université Lyon 1, Lyon 69367, France.
Boulant
Steeve
S
Medical Research Council Virology Unit, Glasgow G11 5JR, Scotland, United Kingdom.
Walic
Marine
M
Unité des Hépacivirus et Immunité Innée and Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France.
Andréo
Ursula
U
Unité des Hépacivirus et Immunité Innée and Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France.
Goueslain
Lucie
L
Institut de Biologie de Lille (UMR8161), CNRS, Université de Lille I & II, Institut Pasteur de Lille, Lille 59021, France.
Helle
François
F
Institut de Biologie de Lille (UMR8161), CNRS, Université de Lille I & II, Institut Pasteur de Lille, Lille 59021, France.
Mallet
Adeline
A
Plate-Forme de Microscopie Ultrastructurale, Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France.
McLauchlan
John
J
Medical Research Council Virology Unit, Glasgow G11 5JR, Scotland, United Kingdom.
Budkowska
Agata
A
Unité des Hépacivirus et Immunité Innée and Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France. Electronic address: agata.budkowska@pasteur.fr.
D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000502:physiology* / Q000648:ultrastructure; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*
D019590:Nucleocapsid Proteins; D014404:Tubulin