Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.
The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.
Angus
Allan G N
AG
MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, UK.
Dalrymple
David
D
Boulant
Steeve
S
McGivern
David R
DR
Clayton
Reginald F
RF
Scott
Martin J
MJ
Adair
Richard
R
Graham
Susan
S
Owsianka
Ania M
AM
Targett-Adams
Paul
P
Li
Kui
K
Wakita
Takaji
T
McLauchlan
John
J
Lemon
Stanley M
SM
Patel
Arvind H
AH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D016174:Hepacivirus / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*; D014779:Virus Replication
D034741:RNA, Small Interfering; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; C119332:DDX3X protein, human; D053487:DEAD-box RNA Helicases