Selection of different 5' untranslated region hepatitis C virus variants during post-transfusion and post-transplantation infection.

BACKGROUND: Hepatitis C virus (HCV) translation is initiated in a cap-independent manner by an internal ribosome entry site (IRES) located within the 5' untranslated region (5'UTR). Sequence changes in this region could affect translation efficiency and presumably viral replication. AIM: To determine translation efficiency of 5'UTR variants developing during post-transfusion hepatitis C in two immunocompetent subjects and in two immunosuppressed liver recipients with recurrent HCV. METHODS: Sequential samples were screened for 5'UTR changes by single-strand conformation polymorphism followed by cloning and sequencing whenever band pattern suggested sequence changes. 5'UTR variants were tested for IRES activity using a bicistronic dual luciferase expression plasmid transfected into HepG2 and Huh7 cell-lines. RESULTS: In the transfused patients, translation efficiency of 5'UTR variants from early post-transfusion samples was 5.1- to 13.7-fold higher than that of predominant variants found in late follow-up samples. Post-transplant variants in the other two patients had 2.6- to 5.9-fold higher translation efficiency than those present only in pretransplant samples. CONCLUSION: In the immunocompetent host there may be selection of low translation efficiency HCV variants over the course of infection. However, in immunosuppressed subjects the opposite seems to be true as low translation efficiency variants are superseded by high translation efficiency variants.

Mouse and frog violate the paradigm of species-specific transcription of ribosomal RNA genes.

Transcription of ribosomal RNA genes by RNA polymerase I is generally accepted as being highly species specific, a conclusion based on numerous reports that rRNA genes of one species are not transcribed by factors of even closely related species. It thus was striking to find that cloned rDNA from the frog Xenopus laevis is specifically transcribed in extracts prepared from mouse cells. The data in this paper demonstrate that this heterologous transcription is due to a normal initiation process and not to a fortuitous event. Transcription of Xenopus rDNA in the mouse cell extract is directed by the same large promoter (residue-141 to +6) that is utilized to promote the synthesis of frog rRNA in homologous Xenopus systems. Moreover, the same factors of the mouse cell extract that transcribe the homologous mouse rDNA also catalyze transcription from the X. laevis rDNA promoter. We conclude that polymerase I transcriptional machinery does not evolve as rapidly as prior studies would suggest.

Transcription of Xenopus ribosomal RNA genes by RNA polymerase I in vitro.

We have developed an in vitro system in which RNA polymerase I accurately transcribes Xenopus ribosomal RNA (rRNA) genes. Cloned Xenopus laevis ribosomal DNA (rDNA) is incubated in a homogenate of manually isolated Xenopus borealis oocyte nuclei. The resultant RNA is analyzed by an S1 nuclease mapping procedure specific for the initiation region of X. laevis rRNA. We show that transcription initiates in vitro on the cloned X. laevis rDNA at the in vivo initiation site. In addition, the dinucleotide ApG significantly stimulates transcription in vitro by acting as a sequence-specific primer. The ApG stimulation is dependent on the template concentration and independent of the ribonucleoside triphosphate levels suggesting that the primer circumvents or augments an initiation-specific factor. We have also investigated transcription of Xenopus rDNA in a mouse cell extract. This heterologous extract can catalyze accurate initiation at a low level. However, under different ionic conditions, this extract transcribes X. laevis rDNA approximately 100-fold more efficiently, but synthesis initiates 4 nucleotides upstream from the in vivo initiation site. There is strong sequence conservation between this start site, the in vivo X. laevis start site, and the in vivo mouse start site suggesting that these sequences are important in RNA polymerase I initiation and that species specificity for RNA polymerase I transcription may not be as great as suggested earlier.