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. 2006 Nov;80(22):11343-54.
doi: 10.1128/JVI.02072-05. Epub 2006 Sep 13.

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Haekyung Lee  1 , Ying Liu, Edison Mejia, Aniko V Paul, Eckard Wimmer
Affiliations

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Haekyung Lee et al. J Virol. 2006 Nov.

Abstract

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

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Figures

FIG. 1.
FIG. 1.
Genomic organization of HCV and of its subgenomic replicon. (A) Genomic structure of full-length HCV RNA. The single-stranded RNA genome of HCV is divided into the 5′ NTR (single line), the polyprotein coding region (boxed), and the 3′ NTR (single line). The polyprotein coding region is divided into structural and nonstructural regions. The NS5B domain, shown enlarged, contains three functional motifs: the catalytic domain (open box), the regulatory motif (gray box), and the membrane anchor (hatched box). (B) Genomic structure of HCV subgenomic replicon RNA. The single-stranded RNA genome of the subgenomic replicon (30) contains the HCV 5′ NTR (single line), the first 16 codons encoding the HCV core protein (ΔC, gray box), the neomycin phosphotransferase gene (NEO; open box), the EMCV IRES (single line), the nonstructural protein coding regions of HCV from NS3 through NS5B (open box), and the 3′ NTR (single line).
FIG. 2.
FIG. 2.
Mutational analyses of some conserved amino acids in the HCV NS5B C-terminal transmembrane domain. (A) NS5B is divided into three functional motifs. The catalytic domain (open box) of NS5B contains the N-terminal 530 amino acids of the protein, which is followed by the noncatalytic region (aa 530 to 591) at the C terminus. Highly conserved amino acids are shown as boldface letters. Underlined amino acid residues correspond to the transmembrane domain of NS5B (hatched box). Negative numbers above the amino acid sequences represent the amino acid positions counted backward from the C-terminal end of the NS5B protein. (B) Effect of point mutations in the NS5B transmembrane domain on the colony-forming efficiency of the subgenomic replicon. (a) Amino acid sequences of the transmembrane domains of wt and mutant NS5B proteins. (b and c) In vitro RNA transcripts of the wt and mutant replicon constructs were transfected into Huh-7 cells, and the colony-forming efficiency of the replicons was measured as described in Materials and Methods. (C) (a) Effect of the R591A mutation in NS5B on transient replication. Huh-7 cells were transfected with the specified luciferase replicons, and luciferase activities were determined in lysates of cells harvested 4 and 72 h after transfection. The 4-h value (not shown) was used to correct for different transfection efficiencies. The 72-h value of luc-5.1 (7.2 × 103 units) was set as 100%. Data are means of three independent experiments. (b) Western blot analysis of cytoplasmic extracts from naïve and luciferase replicon-transfected Huh-7 cells. Western blot analysis was carried out as described in Materials and Methods with polyclonal antibody to NS5B. Lane 1, luc-5.1-transfected cells; lane 2, luc-R591A-transfected cells; lane 3, luc-GAA-transfected cells; lane 4, naïve Huh-7 cells.
FIG. 3.
FIG. 3.
The C-terminal hydrophobic anchor sequence of poliovirus 3A is functional in the HCV NS5B C terminus. (A) Amino acid sequences of the wt and mutant NS5B C-terminal transmembrane domains (hatched box) in the subgenomic replicons. The catalytic domain of NS5B is shown with an open box and the regulatory domain with a gray box. The foreign amino acid sequences from poliovirus 3A are underlined. The entire poliovirus polyprotein and the hydrophobic domain of poliovirus 3A protein are shown with an open box and a dotted box, respectively. The positively charged amino acids (R and K) located at both ends of the hydrophobic domains of HCV NS5B and poliovirus 3A are marked in bold. (B) In vitro RNA transcripts of the wt and mutant replicon constructs were transfected into Huh-7 cells, and the colony-forming efficiencies of the replicons were measured 3 weeks after transfection. The colony-forming efficiencies of the mutant replicons are compared with that of the wt replicon.
FIG. 4.
FIG. 4.
Subcellular localization of wt NS5B and of its variants in Huh-7 cells. (A) Amino acid sequences of the wt and mutant NS5B C-terminal transmembrane domains. The foreign amino acid sequences derived from poliovirus 3A are underlined. The positively charged amino acids located at both ends of the hydrophobic domains of HCV NS5B and poliovirus 3A are marked in bold. NS5B(CΔ21) represents a NS5B protein lacking the C-terminal 21 amino acids. (B) The wt and mutant NS5B proteins were transiently expressed in Huh-7 cells and detected by an indirect immunofluorescence assay with monoclonal antibody 5B-12B7 as described in Materials and Methods.
FIG. 5.
FIG. 5.
Efficient replication of HCV mutant replicons MT(3A-II) and MT(SL-IV) in the established cell lines. (A) Measurement of HCV RNA levels by real-time PCR. Cells from three established cell lines were harvested, and the total RNAs were purified and used for real-time RT-PCR analyses. The quantity of HCV RNA was normalized with that of GAPDH RNA. (B) Agarose gel electrophoresis of the final PCR products obtained by real-time RT-PCR. The PCR products were analyzed by 2% native agarose gel electrophoresis. Lane No, RT-PCR with no RNA; lanes 1pg and 10pg, RT-PCRs with 1 pg and 10 pg of in vitro-transcribed HCV replicon RNA, respectively; lane M, DNA molecular weight marker; lane 1, naïve Huh-7 cells; lane 2, the parental HCV subgenomic replicon (NK5.1); lane 3, MT(3A-II); and lane 4, MT(SL-IV).
FIG. 6.
FIG. 6.
α-Helix projections of HCV NS5B, wt and mutant PV 3A, and BVDV NS5B sequences. The following amino acid sequences were used drawing the helical wheels: HCV NS5B (aa 571 to 588), PV 3A (aa 61 to 78), the PV 3A TT/KK mutant (aa 61 to 78), and BVDV NS5B (aa 699 to 716). The circles are colored according to the hydrophobic character of the residues (39).
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References

    1. Adachi, T., H. Ago, N. Habuka, K. Okuda, M. Komatsu, S. Ikeda, and K. Yatsunami. 2002. The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase. Biochim. Biophys. Acta 1601:38-48. - PubMed
    1. Aizaki, H., K. J. Lee, V. M. H. Sung, H. Ishiko, and M. M. C. Lai. 2004. Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324:450-461. - PubMed
    1. Behrens, S.-E., L. Tomei, and R. de Francesco. 1996. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15:12-22. - PMC - PubMed
    1. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-1974. - PubMed
    1. Brass, V., E. Bieck, R. Montserret, B. Wolk, J. A. Hellings, H. E. Blum, F. Penin, and D. Moradpour. 2002. An amino-terminal amphipathic α-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 277:8130-8139. - PubMed

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