In such cells, the dot-like structures harbor the viral nonstructural proteins and replicating RNA and, therefore, represent replication complexes, which appear as membranous webs by electron microscopy (16)

In such cells, the dot-like structures harbor the viral nonstructural proteins and replicating RNA and, therefore, represent replication complexes, which appear as membranous webs by electron microscopy (16). Open in a separate window FIG. transcription-PCR (RT-PCR) from RNA purified from these clones, followed by sequence analysis. All clones that were positive Benzamide for the FLAG epitope by immunoblot contained an insert close to or within a serine-rich region in the C-terminal domain of NS5A. The one clone that was negative for NS5A-FLAG by immunoblot contained no FLAG insert in NS5A/5B when examined by RT-PCR and sequence analysis. Three replicons contained an insert after amino acid position 2356 (aa 384 of NS5A), and 10 replicons contained an insert after amino acid position 2390 (aa 418 of NS5A). HCV replicons harboring GFP insertions in the C-terminal domain of NS5A are viable. The GFP coding sequence was inserted into the two FLAG-permissive sites in replicon constructs with cell culture adaptive changes in NS3 (E1202G Rabbit polyclonal to KCTD19 and T1280I) and NS4B (K1846T) (GIT) (29) or in NS5A (S2204I) (5) (Fig. ?(Fig.1).1). RNA was in vitro transcribed from these plasmid templates and electroporated into Huh-7.5 cells, followed by G418 selection. Three weeks later, G418-resistant colonies were pooled and expanded or stained with crystal violet, as shown in Fig. ?Fig.2.2. Each of the four different constructs was viable, albeit with different colony formation efficiencies. Counting of G418-resistant colonies resulting from three independent electroporation experiments revealed a colony formation efficiency of about 1 CFU/ng of replicon RNA for GIT/5A-GFP-1, 0.1 CFU/ng for I/5A-GFP-1, 10 CFU/ng for GIT/5A-GFP-6 and I/5A-GFP-6, and 250 CFU/ng for the unmodified parental GIT and S2204I replicons. Thus, replicons harboring the GFP insertion after aa 2390 (GIT/5A-GFP-6 and I/5A-GFP-6) were 10-fold (compared to GIT/5A-GFP-1) or 100-fold (compared to I/5A-GFP-1) more efficient in initiating HCV RNA replication than the constructs with GFP inserted after aa 2356. However, even for I/5A-GFP-1, G418-resistant cell populations or individual clones could be easily expanded, particularly if the G418 concentration was reduced to 400 g/ml (data not shown). Efficiency of the parental replicon constructs GIT and S2204I was about 25-fold higher than that of the constructs harboring the GFP-6 insertion. As expected, the pol? control constructs yielded no G418-resistant colonies. Open in a separate window FIG. 2. HCV replicons harboring GFP insertions in the Benzamide 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. Taken together, initiation of HCV RNA replication by replicon constructs harboring a GFP insertion in NS5A was surprisingly efficient. In good accordance with the different number of clones identified in the initial screen for permissive insertion sites with the FLAG epitope sequence as an insert (3 of 14 after aa 2356 and 10 of 14 after aa 2390), the more C-terminal insertion site was more tolerant of the GFP insert. GFP is retained in NS5A during RNA replication. Western blot analyses of I/5A-GFP-6 and S2204I replicon cells at passage 10, as well as of naive Huh-7.5 cells as negative control, were performed to investigate whether the NS5A-GFP fusion protein was stable during HCV RNA replication. As shown in Fig. ?Fig.3,3, a band of the expected molecular mass of 85 kDa, corresponding to the NS5A-GFP fusion protein, was detected in I/5A-GFP-6 cells by using both MAbs directed against NS5A and against GFP. The unmodified NS5A protein of 56 to 58 kDa was detected in S2204I replicon cells. In addition, correctly processed NS3, NS4B, and NS5B proteins of the expected molecular masses of 70, 27, and 68 kDa, respectively, were detected in both I/5A-GFP-6 and S2204I cells. Analogous findings were obtained for the other replicon constructs. The phosphorylation status of NS5A-GFP was not investigated because replicon constructs harboring the adaptive changes K1846T (present in the GIT constructs) or S2204I (present in the Benzamide I constructs) are impaired for hyperphosphorylation (15). Open in a separate window 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.