Replication and IRES-dependent translation are both affected by core coding sequences in subgenomic GB virus B replicons

Abstract

The yield of G418-resistant Huh7 cell clones bearing subgenomic dicistronic GB virus B (GBV-B) is significantly affected by the insertion of a portion of the viral core gene between the GBV-B 5' untranslated region and the exogenous neomycin phosphotransferase selector gene (A. De Tomassi, M. Pizzuti, R. Graziani, A. Sbardellati, S. Altamura, G. Paonessa, and C. Traboni, J. Virol. 76:7736-7746, 2002). In this report, we have dissected this phenomenon, examining the effects of the insertion of core sequences of different lengths on GBV-B IRES-dependent translation and RNA replication by using experimental approaches aimed at analyzing these two aspects independently. The results achieved indicate that an enhancement of translation efficiency does occur and that it correlates with the length of the inserted core sequences. Interestingly, the insertion of these sequences also has a direct similar effect on the efficiency of replication of the GBV-B replicon. These results suggest that in GBV-B replicon RNA and potentially in the complete viral genome, the core coding sequences not only are part of the IRES but also take part in the replication process, independently of the presence of the corresponding whole protein.

FIG. 1.

Schematic representation of GBV-B neo-Rep and bla-Rep constructs. The nucleotide sequences below the drawing show the boundary between the GBV-B 5′-UTR and the neo or bla gene. The portion belonging to the GBV-B 5′-UTR is underlined, that representing translated GBV-B sequences is shown in bold type, the sequence corresponding to a PmeI restriction site is shown in italic type, and the start Met of either neomycin phosphotransferase or β-lactamase is shown in plain type. The translated sequences are organized in nucleotides triplets, and the corresponding amino acids are indicated below each cognate nucleotide sequence.

FIG. 2.

Effect of the insertion of core coding sequences of different lengths on the colony formation efficiency of GBV-B neo-Rep constructs. (A) Direct visualization of G418-resistant colonies produced by transfecting the same amounts of neo-Rep RNAs into Huh7 cells. A neo-Rep-GAA mutant, which is unable to replicate, was used as a control. After the formation of the colonies, the plates were fixed and colored with crystal violet. (B) Graphic representation of the efficiency of G418-resistant colony formation. Data are reported as ratios of the number of colonies produced by each transfected RNA to the number of colonies obtained with neo-RepA RNA.

FIG. 2.

Effect of the insertion of core coding sequences of different lengths on the colony formation efficiency of GBV-B neo-Rep constructs. (A) Direct visualization of G418-resistant colonies produced by transfecting the same amounts of neo-Rep RNAs into Huh7 cells. A neo-Rep-GAA mutant, which is unable to replicate, was used as a control. After the formation of the colonies, the plates were fixed and colored with crystal violet. (B) Graphic representation of the efficiency of G418-resistant colony formation. Data are reported as ratios of the number of colonies produced by each transfected RNA to the number of colonies obtained with neo-RepA RNA.

FIG. 3.

Effect of core coding sequences on GBV-B replication efficiency. Amounts of RNAs were measured by TaqMan reactions 3 days after transfection of cB76.1/Huh7 cells with bla-Rep RNAs in the absence and in the presence of IFN-α. The amount of RNA detected in each transfection is reported relative to that obtained with a nonreplicating control.

FIG. 4.

Effect of core coding sequences on GBV-B IRES-mediated translation in vivo. cB76.1/Huh7 cells were transfected with the four bla-Rep-GAA RNAs, stained 4 h posttransfection as described in Materials and Methods, observed with a ×10-magnification microscope lens, and photographed with UV light. The cells stained blue contain detectable β-lactamase levels; the cells stained green are background nontransfected cells or cells producing β-lactamase amounts below the detection threshold. The bla-Rep RNA used for each transfection is specified for each panel.

FIG. 5.

Effect of core coding sequences on GBV-B IRES-mediated translation in vitro. Autoradiograms of two sodium dodecyl sulfate-polyacrylamide gels showing the analysis of in vitro-translated bla-Rep (left) and neo-Rep (right) RNAs. The NS3 protein product of each autoradiogram is indicated. Brackets beside the autoradiograms indicate the sections of the gels in which the four reporters of each series, bearing N-terminal fusion fragments of increasing lengths, migrate. The relative amounts of the GBV-B IRES-dependent translation products, calculated by densitometric measurement and normalization to the EMCV IRES-dependent NS3 protein internal control, are reported in the text and correspond to the means for two independent experiments.