Protein-protein interactions between hepatitis C virus nonstructural proteins

Abstract

Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

FIG. 1.

(A) Immunoblot of glutathione-Sepharose-immobilized GST-NS2, GST-NS4B, GST-NS5A, GST, and GST-CD81. Bead-bound bacterially expressed GST-NS fusion proteins (50 μl), as well as GST and GST-CD81 diluted to 1:100, were analyzed by SDS-PAGE followed by immunoblotting with anti-GST antibodies. (B) Autoradiogram from SDS-PAGE analysis of in vitro NS translation products. [³⁵S]methionine-labeled NS proteins (1 μl) were subjected to SDS-PAGE and autoradiography. The positions of molecular size standards are indicated, in kilodaltons.

FIG. 2.

GST pull-down analysis of NS-NS interactions. Each panel shows an autoradiogram from a SDS-PAGE analysis of in vitro-translated NS products selected by binding to the proteins given above the lanes Glutathione-Sepharose-bound GST-CD81 and GST (as controls), GST-NS2, GST-NA4B, and GST-NS5A (50 μl) were incubated with equal amounts of [³⁵S]methionine-labeled in vitro NS translation products, indicated below the panels. Arrows at the right of each panel indicate positions of GST-NS-bound NS proteins. Positions of molecular size standards, in kilodaltons, are shown at the left of each panel.

FIG. 3.

Summary of the GST pull-down assay results. The grid shows strong (•) and weak (○) interactions in the GST pull-down assay. Cells outlined with a boldface box contain new data.

FIG. 4.

In vitro coimmunoprecipitation of NS proteins. In vitro-translated but nonlabeled NS2, NS3, NS4A, NS4B, NS5A, and NS5B were incubated with equal amounts of each [³⁵S]methionine-labeled NS protein to allow partners to interact. Each interaction mixture was immunoprecipitated with rabbit PAbs to NS2, NS3, NS4B, and NS5B or mouse MAbs against NS4A and NS5A (shown by the “@” symbol). Positions of molecular size standards, in kilodaltons, are shown at the left of each panel.

FIG. 5.

Summary of the GST pull-down assay and in vitro coimmunoprecipitation results. The grid shows strong (•) and weak (○) interactions demonstrated in the GST pull-down assay and coimmunoprecipitation interactions (hatched triangle). Cells outlined with a boldface box contain new NS-NS interactions.

FIG. 6.

Identification of NS-NS protein interactions by using the yeast two-hybrid system. (A to F) Yeast strain AH109 was cotransformed by the indicated combinations of plasmids encoding AD-NS (at the top) and BD-NS (at left). Cotransformants were spotted onto plates lacking Leu and Trp, lacking Leu, Trp, and His, or lacking Leu, Trp, and Ade, as indicated below the panels. Growth on plates lacking His or Ade is indicative of an interaction between the indicated AD fusion and BD fusion proteins. Empty vectors (v) pGADT7 (encoding GAL4 AD) and pGBKT7 (encoding GAL4 BD) (Clontech) were used as negative controls. (G) Summary of the NS-NS protein interactions in yeast. For each NS protein combination, growth on culture plates lacking His and/or Ade is represented by a square, as indicated at the right of the chart.

FIG. 7.

NS protein expression in Huh-7 cells. Lysates corresponding to 8 × 10⁵ Huh-7 cells infected with AdIV1053 or AdIV1043 (as a control) (MOI, 75) were harvested at 65 h postinfection, loaded onto a gel, separated by electrophoresis, blotted, and probed with rabbit PAbs to NS2, NS3, NS4B, and NS5B and mouse MAbs against NS5A.

FIG. 8.

Coimmunoprecipitation of NS proteins expressed in Huh-7 cells. Lysates corresponding to 8 × 10⁵ Huh-7 cells infected with AdIV1053 or AdIV1043 (as a control) (MOI, 75) were harvested at 65 h postinfection. NS protein complexes were coimmunoprecipitated (IP) with rabbit PAbs to NS3 (A, D, and F) or NS4B (B and E) and mouse MAbs against NS5A (C). Coimmunoprecipitated NS proteins were detected by SDS-PAGE and immunoblotting with antibodies to GST-NS2 (A), NS3 (B and C), NS5A (D and E), or NS5B (F). Positions of molecular size standards, in kilodaltons, are shown at the left of each panel.

FIG. 9.

Colocalization of NS proteins expressed in Huh-7 cells. Huh-7 cells (3 × 10⁴) infected with AdIV1053 (MOI, 75) were processed for double immunolabeling at 65 h postinfection as described in Materials and Methods with rabbit PAbs against NS2 (A, B, and E), NS4B (C, D, and F), or NS5B (G and H) and with mouse MAbs against NS3 (A), NS4A (F and G), NS4B (B), NS5A (C, E, and H), or NS5B (D). Bound primary antibodies were revealed with Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies, and slides were analyzed by confocal laser scanning microscopy.

FIG. 10.

Compilation of GST pull-down assay results, in vitro and ex vivo coimmunoprecipitation results, and two-hybrid system results. Results are depicted as follows: • and ○, strong and weak interaction, respectively, in the GST pull-down assay; , in vitro coimmunoprecipitation; ,  and □, interaction, weak interaction (i.e., directional interaction and/or interaction observed only once on medium lacking His), and no interaction in the two-hybrid system, respectively; and ◊, coimmunoprecipitation and no coimmunoprecipitation in Huh-7 cells, respectively. Cells outlined with a boldface box contain new data.