Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes

Abstract

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

FIG. 1.

Replicon constructs. (A) Sites and amino acid sequences of the GFP insertions in NS5A. (B) Replicon constructs. Cell culture adaptive mutations in NS3 and NS4B (GIT constructs) or NS5A (S2204I constructs) are marked by circles. The pol⁻ control constructs harbor an inactivating GDD→AAG mutation in the NS5B RNA-dependent RNA polymerase.

FIG. 2.

HCV replicons harboring GFP insertions in the C-terminal region of NS5A are viable. RNA was in vitro transcribed from constructs GIT/5A-GFP-1, I/5A-GFP-1, GIT/5A-GFP-6, I/5A-GFP-6, and S2204I, as well as pol⁻/5A-GFP-1 and pol⁻/5A-GFP-6, and electroporated into Huh-7.5 cells, followed by plating into 100-mm-diameter dishes at 6 × 10⁵, 6 × 10⁴, and 6 × 10³ cells per dish and G418 selection as described in Materials and Methods. G418-resistant colonies were stained with crystal violet after 3 weeks.

FIG. 3.

GFP is retained in NS5A during RNA replication. Lysates of Huh-7.5 cells harboring the replicon constructs I/5A-GFP-6 (5A-GFP) and S2204I, as well as lysates of naive Huh-7.5 cells, were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with MAbs 1B6 against NS3, 4b-52 against NS4B, 11H against NS5A, 5B-3B1 against NS5B, or JL-8 against GFP. About 100 μg of cellular protein was loaded per lane. Molecular mass standards in kilodaltons are indicated on the left.

FIG. 4.

NS5A-GFP can be directly visualized by fluorescence microscopy. Huh-7.5 cells harboring the constructs GIT/5A-GFP-6 (A) and I/5A-GFP-6 (B) were analyzed by fluorescence microscopy at 5 days postseeding. (C) Cells harboring the parental S2204I replicon without GFP insertion served as the negative control.

FIG. 5.

NS5A-GFP colocalizes with other HCV nonstructural proteins. Huh-7.5 cells harboring replicon construct I/NS5A-GFP-6 were stained with MAb 11H against NS5A (A) or MAb 1B6 against NS3 (B), followed by CLSM as described in Materials and Methods. Nuclei were counterstained with TO-PRO-3 iodide.

FIG. 6.

NS5A-GFP colocalizes with nascent HCV RNA. I/5A-GFP-1 replicon cells were metabolically labeled with BrUTP in the presence of actinomycin D, followed by CLSM. (A) GFP fluorescence. (B) Detection of newly synthesized, BrU-labeled viral RNA with a MAb against BrdU and a Cy3-conjugated secondary antibody. (C) The overlay demonstrates colocalization (yellow) of the NS5A-GFP fusion protein and nascent viral RNA. The border and the nucleus of a single cell are highlighted by thick and thin lines, respectively. Negative controls were identically treated naive Huh-7 cells. Comparable results were obtained in cells harboring replicons with the GIT adaptive background.

FIG. 7.

Ultrastructure of GFP replicon cells and immunogold detection of GFP and HCV nonstructural proteins on the membranous web. (A) Huh-7.5 cells harboring replicon construct I/5A-GFP-1 were analyzed by EM at 5 days postseeding. A membranous web is marked by the arrows. Bar, 500 nm. ER, endoplasmic reticulum; G, Golgi apparatus; M, mitochondria; N, nucleus. The same cells were analyzed by double-label IEM with MAb JL-8 against GFP and the NS5A-specific polyclonal antiserum WU144, followed by 10- and 15-nm gold-conjugated secondary antibodies, respectively (B), or an anti-GFP polyclonal antiserum and MAb 1B6 against NS3, followed by 15- and 10-nm gold-conjugated secondary antibodies, respectively (C). GFP and HCV nonstructural proteins are found almost exclusively on the membranous web. Bars, 200 nm.