Student’s t-test (unpaired) was performed by using GraphPad Prism version 6 software to determine the significance in differences between paired values. A P value less than 0.05 was considered statistically significant.
Supporting information
S1 Fig. The residue interaction network showing the non-covalent residue interactions within two different NS5A-DI dimer structures. We used experimental structure informa-tion from the Protein Databank RCSB PDB [74] for 3D structure analysis and computed residue-interaction networks (RINs) for PDB-1ZH1 (A) and PDB-3FQM (B) by using the RINerator tool [75]. To this end, hydrogens are first added to the 3D protein structure by using the Reduce tool [76]. Then contacts on the van-der-Waals (vdW) surface of each atom
Roles of HCV NS5A dimer interface residues in HCV replication
are sampled by the Probe tool [77] and are finally summarized into residue interactions by RINerator. In the resulting network, nodes represent protein residues and edges indicate non-covalent residue interactions between these residues. The following interaction types are iden-tified: van-der-Waals contacts (cnt), hydrogen bonds (hbond), overlaps of van-der-Waals radii (ovl), and combined (any of the previous three). We also distinguish between two types of interacting atoms: main chain (mc) and side chain (sc). The RINs are loaded and visualized in Cytoscape (http://www.cytoscape.org) by using the RINalyzer and structureViz2 apps [78].
Residues from different NS5A monomers are colored in yellow and salmon and designated as
“A” and “B”, respectively.
(TIF)
S2 Fig. The effects of 112E, 122K, 148D and 148R mutations on JFH1 NS5A self-interac-tion. JFH1 NS5A self-interaction was determined by a checkmate mammalian two-hybrid assay. Statistical analysis was performed by using student’s t-test as described in Materials and Methods.
(TIF)
S3 Fig. The effects of DEYN, 146A or 146D NS5A mutations on H77 or JFH1 NS5A self-interaction. The effects of NS5A-DEYN (D316E/Y317N) mutations (A) or 146A and 146D mutations (B) on H77 or JFH1 NS5A self-interaction as determined by a checkmate mamma-lian two-hybrid assay.
(TIF)
S4 Fig. Detection of NS5A-CypA interaction only in GAL4/BD-CypA and VP16/AD-NS5A pair configuration of checkmate assay. CypA and NS5A interaction was determined by a checkmate mammalian two-hybrid assay. Statistical analysis was performed by using student’s t-test as described in Materials and Methods.
(TIF)
S5 Fig. Effects of NS5A dimer interface mutations in JFH1/QL replication and infectivity.
JFH1/QL was generated by introducing cell culture adaptive mutation in NS3 residue Q221L [44]. (A) NS5A western blot analysis of JFH1/QL wt and NS5A dimer interface mutants, which indicates the defective replication of 36A and 112A mutants and also the hyperpho-sphorylation impairment in 37A, 38A and 148A mutants. (B) Left and right panels show the virus titration results by using cell lysates (intracellular) and cell culture supernatants (extracel-lular), respectively, at different time points post-electroporation of JFH1/QL RNA with or without indicated mutations. (C) Percentage of intracellular virus titers per total (intracellular plus extracellular) virus titer.
(TIF)
S6 Fig. JFH1 NS5A hyperphosphorylation and NS5A-core interaction detected following expression of HCV polyproteins. (A) Western blot analysis of NS5AYFPfollowing expression of full length HCV polyprotein derived from HJ3-5/NS5AYFPby using T7 based expression system composed of pTM1 vector ([79], kindly provided by Dr. Bernard Moss at the National Institute of Health, USA) and T7-Lunet cells ([80], kindly provided by Dr. Bartenschlager at the University of Heidelberg, Germany). (B) GFP immunoprecipitation (IP) followed by west-ern blot analysis to detect NS5A and core interaction. Shown at the bottom is the relative NS5A and core co-immunoprecipitation (co-IP) efficiency.
(TIF)
S7 Fig. NS5A/36V mutations on H77 NS5A self-interaction and NS5A-CypA interaction.
The effects of NS5A/36V mutations on H77 NS5A self-interaction (A) and NS5A-CypA
interaction (B) as determined by a checkmate mammalian two-hybrid assay.
(TIF)
Acknowledgments
We thank Stanley M. Lemon for the H77D clone, Takaji Wakita for the JFH1 clone, Bernard Moss for the pTM1 vector, Ralf Bartenschlager for T7-Lunet cell line and Charles M. Rice for Huh7.5 cell line used in this study.
Author Contributions
Conceptualization: Saravanabalaji Shanmugam, Christoph Welsch, MinKyung Yi.
Formal analysis: Christoph Welsch.
Funding acquisition: Christoph Welsch, MinKyung Yi.
Investigation: Saravanabalaji Shanmugam, Alyssa K. Nichols, Dhanaranjani Saravanabalaji, MinKyung Yi.
Methodology: Saravanabalaji Shanmugam, Alyssa K. Nichols, Dhanaranjani Saravanabalaji, Christoph Welsch, MinKyung Yi.
Project administration: MinKyung Yi.
Resources: MinKyung Yi.
Supervision: MinKyung Yi.
Writing – original draft: Saravanabalaji Shanmugam, Christoph Welsch, MinKyung Yi.
Writing – review & editing: Saravanabalaji Shanmugam, Alyssa K. Nichols, Christoph Welsch, MinKyung Yi.
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