Mutation Master: Profiles of substitutions in hepatitis C virus RNA of the core, alternate reading frame, and NS2 coding regions

Abstract

The RNA genome of the hepatitis C virus (HCV) undergoes rapid evolutionary change. Efforts to control this virus would benefit from the advent of facile methods to identify characteristic features of HCV RNA and proteins, and to condense the vast amount of mutational data into a readily interpretable form. Many HCV sequences are available in GenBank. To facilitate analysis, consensus sequences were constructed to eliminate the overrepresentation of certain genotypes, such as genotype 1, and a novel package of sequence analysis tools was developed. Mutation Master generates profiles of point mutations in a population of sequences and produces a set of visual displays and tables indicating the number, frequency, and character of substitutions. It can be used to analyze hundreds of sequences at a time. When applied to 255 HCV core protein sequences, Mutation Master identified variable domains and a series of mutations meriting further investigation. It flagged position 4, for example, where 90% or more of all sequences in genotypes 1, 2, 4, and 5, have N4, whereas those in genotypes 3, 6, 7, 8, 9, and 10 have L4. This pattern is noteworthy: L (hydrophobic) to N (polar) substitutions are generally rare, and genotypes 1, 2, 4, and 5 do not form a recognized super family of sequences. Thus, the L4N substitution probably arose independently several times. Moreover, not one member of genotypes 1, 2, 4, or 5 has L4 and not one member of genotypes 3, 6, 7, 8, 9, or 10 has N4. This nonoverlapping pattern suggests that coordinated changes at position 4 and a second site are required to yield a viable virus. The package generated a table of genotype-specific substitutions whose future analysis may help to identify interacting amino acids. Three substitutions were present in 100% of genotype 2 members and absent from all others: A68D, R74K, and R114H. Finally, this study revealed thatARFP, a novel protein encoded in an overlapping reading frame, is as conserved as conventional HCV proteins, a result supporting a role for ARFP in the viral life cycle. Whereas most conventional programs for phylogenetic analysis of sequences provide information about overall relatedness of genes or genomes, this program highlights and profiles point mutations. This is important because determinants of pathogenicity and drug susceptibility are likely to result from changes at only one or two key nucleotides or amino acid sites, and would not be detected by the type of pairwise comparisons that have usually been performed on HCV to date. This study is the first application of Mutation Master, which is now available upon request (http://tandem.biomath.mssm.edu/mutationmaster.html).

Unusual Multiple Recoding Events Leading to Alternative Forms of Hepatitis C Virus Core Protein from Genotype 1b

“…Enzyme Immunoassay-Three synthetic peptides encoded in the ϩ1 ORF of core and predicted to be antigenic were obtained by chemical synthesis: peptide F1 (core (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), NVTPTAAHRTLSSRA), peptide F2 (Core (46 -60), ARLGRLPSGRNLVEG), and peptide F3 (Core (106 -120), GAPQTPGVGRVIWVR). For the enzyme immunoassay, the wells of a microtiter plate were coated with 0.5 g of peptide and incubated with 10 l of human serum and 90 l of diluent at room temperature for 1 h. The wells were washed and subsequently incubated with 100 l of a 1:3,000 dilution of a horseradish peroxidase-conjugated goat antihuman antibody (Pierce).…”

“…6). As shown in this figure, when the peptide used belongs to the amino acid sequence in the ϩ1 ORF located before amino acid 42 (core (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), namely peptide F1), 2 of 10 sera from genotype 1a are reactive, whereas no serum from genotype 1b is reactive. In contrast, when the peptides used belong to the amino acid sequence in the ϩ1 ORF located after amino acid 42 (namely peptides F2 (core (46 -60)) and F3 (core (106 -120))), 3 of 10 sera from genotype 1a and 6 of 10 sera from genotype 1b react with peptide F2, and 3 of 10 sera from genotype 1a and 1 of 10 sera from genotype 1b react with peptide F3.…”

“…These results strengthen the hypothesis that an alternative core protein is expressed in vivo in genotype 1b as a result of a ϩ1 frameshifting. In addition, the absence of reactivity of sera of genotype 1b against peptide F1 (core (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)) indicate that the shift site is downstream of position 25. This supports our finding that the shift site leading to F protein is different in genotypes 1a and 1b (shift site at or near codon 11 for the former and at codon 42 for the latter).…”

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

“…The length of the core11 protein appears to be genotype-specific, varying between 126 and 162 amino acids (162 aa for genotype 1a, 144 aa for 1b, 126 aa for 2a, and 126-155 amino acids for most of the sequences of other genotypes) (19). The core11 amino-acid sequence displays a lower level of conservation than the core protein (50,54,55), but is at least as well conserved as the E1 and NS2 HCV proteins encoded by the main ORF (55). Similar results were obtained in both conventional homology analysis, in which proteins encoded by diverse HCV RNA sequences are compared in pairs to determine the percentage of identical amino acids, and with a new package of data mining tools (Mutation Master) comparing individual sequences in a multiple alignment with a reference sequence (consensus sequence constructed to correct for the overrepresentation of certain genotypes) at each amino acid position.…”

Structural analysis of hepatitis C RNA genome using DNA microarrays

“…The earliest evidence that the HCV core RNA sequence harbours additional functions than coding for a single protein was the observation that the rate of synonymous substitution for the core gene was remarkably lower than that for the other HCV genes (55,56). The limitation in the number of synonymous substitutions in this region has been explained by the possibility of encoding an alternative protein by frame shifting (ARFP) (57,58), and by the existence of internally base-paired stem-loop structures in this region (39). Our results suggest that at least the first three-quarters of the core region contain a number of RNA structural elements that probably lead to structural constraints to nucleotide replacements in this region.…”