Aberrant transcription and post-transcriptional processing of hepatitis C virus non-structural genes in transgenic mice

Abstract

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease worldwide. Since several aspects of the infection remain unresolved, there is a pressing need for a convenient animal model that can mimic the clinical disease and aid the evaluation of treatment strategies. Although some success has been achieved in transgenic approaches for development of rodent models of HCV, transgenic expression of the complete HCV polyprotein or an entire set of the viral non-structural (NS) proteins continues to be a serious challenge. Using northern blot and 5′ rapid amplification of cDNA ends (RACE), we unraveled two possible mechanisms that can impede HCV NS transgene expression in the mouse liver. Several truncated transcripts are produced from alternate transcription start sites along the HCV NS sequence within the murine environment, in vivo. Translation of these shorter transcripts is blocked either by the positioning of a contextual stop codon or through a shift in the reading frame. In addition, the complete NS transcript undergoes trans-splicing through 5′ recombination with a non-transgene-derived, spliced leader sequence that appends a potential stop codon upstream of the translation start. These findings thus demonstrate that HCV NS-derived transgenes are subject to aberrant transcriptional initiation and post-transcriptional processing in the nucleus of a mouse host. Strategies to prevent such aberrant transcription start/RNA processing might be key to the development of a successful HCV transgenic mouse model.

Homologous SV40 RNA trans-splicing

“…This hypothesis is supported by previous descriptions of heterologous trans-splicing that all involved viral transcripts, such as between the HIV-nef pre-mRNA, 56 the adenoviral major late transcript, 17 or a HCV transgenic transcript 57 and cellular sequences or between premRNAs from SV40 and HIV. 56 Functions have not been assigned to the resulting fusion proteins.…”

Viral RNA Trans-Splicing: Chance Event or Product of Evolution?

“…Computational analyses of cDNAs from a gene databank indicated one percent of all sequenced mRNAs to be chimeric [7], some of which might be synthesized by RNA trans-splicing. Up to date several examples of naturally occurring mammalian RNA trans-splicing have been reported [8][9][10][11][12][13][14].…”

“…Though being kinetically less favoured examples of cross-species or species internal heterologous transsplicing were reported. These include trans-splice combinations between two viruses as SV40 and HIV [9], or between cellular RNA sequences and viral RNAs like HIV-nef pre-mRNA [9], the adenoviral major late transcript [11], or a HCV transgenic transcript [14]. The examples reported so far impressively demonstrate a frequent involvement of viral transcripts in transsplicing reactions.…”

“…Computational analyses of cDNAs from a gene databank indicated one percent of all sequenced mRNAs to be chimeric [7], some of which might be synthesized by RNA trans-splicing. Up to date several examples of naturally occurring mammalian RNA trans-splicing have been reported [8][9][10][11][12][13][14].A major difference between cis-and trans-splicing relies in the fact that the efficiency of trans-splicing strongly depends on the probability that two separated RNA precursor molecules encounter each other in the nuclear compartment. This likelihood is maximal, when the two RNA precursors originate from the same transcriptional locus and when they contain antisense binding domains to trigger kind of intron-like bridging between splice sites.…”