Human La protein interaction with GCAC near the initiator AUG enhances hepatitis C Virus RNA replication by promoting linkage between 5' and 3' untranslated regions

Abstract

Human La protein has been implicated in facilitating the internal initiation of translation as well as replication of hepatitis C virus (HCV) RNA. Previously, we demonstrated that La interacts with the HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG within stem-loop IV by its RNA recognition motif (RRM) (residues 112 to 184) and influences HCV translation. In this study, we have deciphered the role of this interaction in HCV replication in a hepatocellular carcinoma cell culture system. We incorporated mutation of the GCAC motif in an HCV monocistronic subgenomic replicon and a pJFH1 construct which altered the binding of La and checked HCV RNA replication by reverse transcriptase PCR (RT-PCR). The mutation drastically affected HCV replication. Furthermore, to address whether the decrease in replication is a consequence of translation inhibition or not, we incorporated the same mutation into a bicistronic replicon and observed a substantial decrease in HCV RNA levels. Interestingly, La overexpression rescued this inhibition of replication. More importantly, we observed that the mutation reduced the association between La and NS5B. The effect of the GCAC mutation on the translation-to-replication switch, which is regulated by the interplay between NS3 and La, was further investigated. Additionally, our analyses of point mutations in the GCAC motif revealed distinct roles of each nucleotide in HCV replication and translation. Finally, we showed that a specific interaction of the GCAC motif with human La protein is crucial for linking 5' and 3' ends of the HCV genome. Taken together, our results demonstrate the mechanism of regulation of HCV replication by interaction of the cis-acting element GCAC within the HCV IRES with human La protein.

Fig 1

Effect of mutation of the GCAC motif in SLIV on HCV RNA functions. (A) Schematic representation of the HCV 5′ UTR showing the position of the mutation incorporated into the SLIV region of the HCV monocistronic subgenomic replicon (33). Nucleotide positions of initiator AUG (iAUG) and the GCAC motif are also marked. (B) Huh7 cells were transfected with either in vitro-transcribed wild-type or mutated HCV monocistronic subgenomic RNA. The cells were harvested at 24 h posttransfection, and HCV RNA (negative-strand) levels were quantified by using semiquantitative RT-PCR. (C) Serial dilutions of the cDNA samples (1:2, 1:4, 1:8, and 1:16) were made. Amplified PCR products were analyzed by electrophoresis on a 2% agarose gel. GAPDH was used as an internal control. “U” denotes undiluted cDNA sample. (D to F) Tagged cDNA RT-PCR (D), quantitative RT-PCR (E), and Western blot for NS5B using anti-NS5B antibody (F) for the experiment performed in panel B. Actin was used as a control to ensure equal loading. (G) Huh7.5 cells were transfected with wild-type or mutated HCV JFH1 RNA. The cells were harvested at 24 h posttransfection, and HCV RNA (negative-strand) levels were quantified by using quantitative RT-PCR. (H) For the experiment performed in panel G, Western blotting for NS5B was done by using anti-NS5B antibody. Actin was used as a control to ensure equal loading.

Fig 2

Effect of mutation of the GCAC motif on HCV RNA stability. (A) Huh7 cells were cotransfected with either in vitro-transcribed wild-type or mutated HCV monocistronic subgenomic RNA and a Renilla luciferase reporter construct. At 24 h posttransfection, a luciferase assay was performed, and the results are represented graphically (“ns” represents values that are not significant). (B) Huh7.5 cells were cotransfected with either in vitro-transcribed wild-type or mutated HCV JFH1 RNA and a Renilla luciferase reporter construct. At 24 h posttransfection, a luciferase assay was performed, and the results are represented graphically. (C) Integrity of in vitro-transcribed wild-type and mutated HCV monocistronic RNAs was checked in a 0.8% formaldehyde agarose gel. (D and E) Huh7 cells were transfected with wild-type or mutated HCV monocistronic subgenomic RNA and treated simultaneously with hemin (100 μM). Total RNA was isolated at 12 h and 24 h posttransfection, and the level of input positive-strand HCV was checked by quantitative RT-PCR using primers corresponding to either the 5′ UTR (D) or the 3′ UTR (E). GAPDH was used as an internal control for normalization [HCV(+) denotes HCV positive strand].

Fig 3

Effect of mutation of the GCAC motif on La interaction with HCV RNA. (A) Huh7 cells were transfected with in vitro-transcribed (positive-sense) wild-type or mutant monocistronic subgenomic RNA and simultaneously treated with hemin (100 μM). Cells were harvested at 24 h posttransfection, and ribonucleoprotein (RNP) complexes were immunoprecipitated by using anti-La antibody, followed by quantitative RT-PCR of the positive-sense RNA extracted from the immunoprecipitated complex. (B) Western blotting was done to verify equal precipitation of La. (C) Huh7 cells were transfected with wild-type or mutated HCV monocistronic subgenomic RNA. The cells were harvested at 24 h posttransfection, and RNA-protein complexes were precipitated by using anti-La antibody, followed by tagged cDNA RT-PCR for HCV negative-strand RNA. IP, immunoprecipitation. (D) La overexpression can rescue the inhibition of HCV RNA replication. Huh7 cells were cotransfected with wild-type or mutant HCV monocistronic subgenomic RNA along with a plasmid construct expressing either La or P4La (mutant La). The cells were harvested at 24 h posttransfection, and negative-strand HCV RNA levels were quantified by quantitative RT-PCR (“C” denotes control, “Wt” denotes wild-type RNA, “Mut” denotes mutated RNA, and “ns” represents values that are not significant). (E) Western blotting was done by using anti-NS5B antibody to check NS5B production levels. (F) La and P4La overexpression was checked by Western blot analysis using anti-La antibody.

Fig 4

Mutation of the GCAC motif affects HCV replication independent of translation. (A) Huh7 cells were transfected with in vitro-transcribed wild-type or mutant pSGR-JFH1/Luc RNA. The cells were harvested at 24 h and 48 h posttransfection, and HCV RNA levels were quantified by using semiquantitative RT-PCR. The schematic above the panel represents the pSGR-JFH1/Luc replicon (35). (B) Luciferase levels were also assayed. (C) Effect of La silencing on HCV replication. Huh7 cells were cotransfected with wild-type or mutant pSGR-JFH1/Luc RNA and siLa (50 nM). The cells were harvested at 24 h and 48 h posttransfection, and HCV RNA levels were quantified by using semiquantitative RT-PCR. (D) Western blotting was done for La by using anti-La antibody to check silencing. Actin was used as a control to ensure equal loading. (E) A luciferase assay was also performed, and the results are represented graphically. (F) Western blotting for NS3 was done using by anti-NS3 antibody to check EMCV IRES-mediated translation. Actin was used as a control to ensure equal loading.

Fig 5

Mutation of the GCAC motif affects interactions between La and NS5B. Huh7 cells were transfected with in vitro-transcribed wild-type or mutated pSGR-JFH1/Luc RNA along with treatment with hemin (100 μM). At 24 h posttransfection, immunoprecipitation was done by using anti-La antibody (A) or anti-NS5B antibody (B), followed by Western blot analysis for La and NS5B. Anti-IgG antibody was used as a negative control for immunoprecipitation. “Control” indicates no transfection.

Fig 6

Effect of exogenous La on HCV replication and translation. (A) Huh7 cells were cotransfected with wild-type or mutated pSGR-JFH1/Luc RNA and a construct expressing La/P4La (mutant La). The cells were harvested at 24 h posttransfection, and replication was checked by semiquantitative RT-PCR (“C” denotes control, “Wt” denotes wild type, and “Mut” denotes mutant). (B) A luciferase assay was also performed to check HCV IRES-mediated translation.

Fig 7

Effect of mutation of the GCAC motif on the translation-to-replication switch. (A and B) Huh7 cells were cotransfected with wild-type (A) or mutant (B) pSGR-JFH1/Luc RNA and a construct expressing NS3^pro or the vector. Total RNA was isolated at different time points, and HCV RNA was quantified by using semiquantitative RT-PCR (left). GAPDH was used as an internal control (right). (C and D) For the experiments detailed in panels A and B, luciferase assays were performed to detect translation levels. The percent luciferase activities were plotted for each reaction at different time points, taking the 6-h time point as 100% (control) (“ns” represents values that are not significant). (E and F) The ratio of percent replication to translation levels in vector-transfected control cells was compared with that of cells expressing NS3^pro and is graphically represented by plotting the ratio of percent replication to translation on the y axis and time on the x axis. (G) For the experiments performed in panels A and B, the RNA protein complex was immunoprecipitated at 18 h posttransfection by using anti-NS5B antibody followed by semiquantitative RT-PCR for HCV negative-strand RNA. (H) Huh7 cells were cotransfected with in vitro-transcribed wild-type or mutated pSGR-JFH1/Luc RNA and a construct expressing NS3. Immunoprecipitation was done at 18 h posttransfection by using anti-La antibody, followed by Western blotting for NS3 by using anti-NS3 antibody. Western blotting for La was done to verify equal precipitation. Anti-IgG antibody was used as a negative control for immunoprecipitation. “Control” represents no transfection. − and + represent “without RNase A treatment” and “with RNase A treatment,” respectively.

Fig 8

The GCAC motif participates directly in enhancing HCV replication. (A and B) Huh7 cells were transfected with in vitro-transcribed wild-type or mutated pSGR-JFH1/Luc RNA, followed by treatment with puromycin (A) or cycloheximide (B) (12 h posttransfection) and detection of HCV negative-strand RNA at different time points (as indicated) by using a two-cycle RNase protection assay (the arrow above the 12-h time point indicates the time point of addition of puromycin or cycloheximide). (C and D) Luciferase assays were performed to check HCV IRES-mediated translation.

Fig 9

Effect of point mutations of the GCAC motif on HCV replication and translation. (A) Huh7 cells were transfected with in vitro-transcribed wild-type or mutated pSGR-JFH1/Luc RNA. The cells were harvested at 24 h posttransfection, and HCV negative-strand RNA levels were quantified by using semiquantitative RT-PCR. GAPDH was used as an internal control (ACCG indicates the same mutant represented in Fig. 1A, whereas G345A, A347C, C348G, A338U, and C324U represent point mutants). (B) One microgram of wild-type and various mutated pSGR-JFH1/Luc RNAs was translated in RRL, and percent luciferase activities were plotted (“ns” represents data that are not significant). (C) Wild-type and mutated (G345A, A347C, and C348G) HCV IRES-Luc RNAs (42) were translated in vitro in RRL, and percent luciferase activities were plotted. (D) In vitro binding of α-³²P-labeled wild-type and mutated HCV IRES RNAs with increasing concentrations of recombinant La was performed by using a UV cross-linking assay. No protein was used as a negative control.

Fig 10

La protein interaction with the GCAC motif promotes 5′-to-3′ linkage. (A) Schematic representation of the 5′-3′ coprecipitation assay carried out with a biotinylated 5′ UTR and a ³²P-labeled 3′ UTR. The location of the GCAC motif is also indicated. (B) Autoradiograph of precipitated RNAs in the absence or presence of increasing amounts of La (25, 50, and 100 ng). BSA (100 ng) was used as an unrelated protein control. (C) Increasing amounts of unlabeled (cold) 5′ UTR and 3′ UTR competed away the binding of the ³²P-labeled 3′ UTR to the biotinylated 5′ UTR (Bio-5′UTR). (D and E) The same assay was performed with wild-type and various indicated mutated 5′-UTR RNAs in the presence of La (50 ng). Nonspecific RNA (unrelated nonviral RNA) that replaced the 5′ UTR in the assay mixture was used as a control.

Fig 11

Model for initiation of HCV replication. La interaction with the GCAC motif within HCV IRES promotes 5′-to-3′ communication in favor of HCV negative-strand synthesis by assisting in the assembly of the replication complex.