Structural analysis of hepatitis C RNA genome using DNA microarrays

Abstract

Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5 0 non-coding region (5 0 NCR), the first threequarters of the core region, the E2-NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions.

Collective properties of evolving molecular quasispecies

“…As an example, both approaches have been successfully applied to the analysis of the very structurally compact internal ribosome entry sites (IRES) elements in viral RNA, and they have allowed the characterization of functionally relevant tertiary interactions [42,43]. Recently, an alternative method of RNA structure analysis has been developed, based on the hybridization of RNA in native conditions to microarrays of complementary DNA oligonucleotides [44,45]. Additionally, the three-dimensional solution structure of different biologically active RNAs can be experimentally determined by means of different approaches, including X-Ray diffraction techniques [46] and Nuclear Magnetic Resonance-based methods [47].…”

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

“…Our data show that an RNAse III-sensitive structure converts the 5′-UTR and 1/3 of the core-coding region into a circular RNA loop, closed by base pairing as shown in Figure 8. The IRES and core-coding regions, in addition to sharing a highly structured character (15,42,43), overlap in several other ways. First, the IRES contains the first 30 bases of the core-coding sequence.…”

The folding of the hepatitis C virus internal ribosome entry site depends on the 3′-end of the viral genome

“…The unbound DNA oligos and the excess of spotting solution were washed by an additional incubation in 2× SSC for 2 min. Then, printed oligos were denatured by baking the microarrays for 2 min in boiling milli-Q water, cooling for 10 s at RT and immediately fixed in ice-cold 100% ethanol as described (42). The microarrays were prehybridized in 6× SSC, 0.5% SDS and 10 µg/µl BSA for 45 min at 42°C.…”