1 Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, Uruguay
2 Cátedra de Hemoterapia, Facultad de Medicina, Av. Italia s/n, Montevideo, Uruguay
3 Instituto de Investigaciones Clínicas, Facultad de Medicina San Fernando, Universidad Nacional Mayor de San Marcos, Parque de la Medicina, Avenida Grau Cuadra 13 s/n, Lima 01, Peru
4 Servicio de Inmunología, Hospital Nacional Edgardo Rebagliati Martins HNERN, Domingo Cueto s/n, Jesús María, Lima 11, Peru
Correspondence
Juan Cristina
cristina{at}cin1.cin.edu.uy
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this work are AJ582128AJ582131 and AJ781117AJ781124.
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INTRODUCTION |
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METHODS |
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RNA extraction and cDNA synthesis and amplification.
HCV RNA was extracted from serum samples (100 µl) by using a QIAamp viral RNA kit (Qiagen) according to the manufacturer's instructions. Extracted RNA was eluted from the columns with 50 µl RNase-free water, and cDNA synthesis and PCR amplification of the core region were carried out as described by Bukh et al. (1994). To avoid false-positive results, the recommendations of Kwok & Higuchi (1989)
were strictly adhered to. Amplicons were purified by using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions.
Sequencing.
The primers used for amplification were also used for sequencing the PCR fragments. All PCR fragments were sequenced in both directions to avoid discrepancies. The sequencing reaction was carried out by using a BigDye DNA sequencing kit on a 373 DNA sequencer apparatus (both from Perkin Elmer).
Sequence analysis.
The amino acid sequences of the core protein, as well as the F protein (obtained from the core gene in the F reading frame), were aligned by using the CLUSTAL W program (Thompson et al., 1994).
Substitution-rate analysis.
Substitution rates along the HCV F protein were measured by using a sliding window. Pairwise nucleotide distances (synonymous and non-synonymous) within each window were estimated by the method of Comeron (1995), as implemented in the computer program K-estimator. For those windows where the method was inapplicable (due to the negative argument of the logarithm), we used the JukesCantor method (Jukes & Cantor, 1969
). The window size used was 30 codons, shifting three codons at a time. The ratios of non-synonymous (dn) to synonymous (ds) substitutions for the core and F proteins were calculated by using data obtained from the computer program SNAP, as implemented by Korber (2000)
.
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RESULTS |
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As shown in Fig. 1, the rates of non-synonymous substitutions were significantly higher than those of synonymous substitutions for all pairwise comparisons, in agreement with previous results (Table 2
). Interestingly, the profiles of synonymous and non-synonymous distances exhibited low covariation. This means that those regions of the F protein that are more divergent at the amino acid level are not more divergent at the synonymous level (see Fig. 1
).
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Distribution of stop codons in the F protein across all HCV genotypes
In order to gain insight into the functionality of the F protein, we studied the distribution of stop codons across all HCV genotypes and subtypes available in the HCV databases. As shown in Table 3, some genotypes, particularly 2 and 3, had more stop codons in their F proteins than subtypes 1a and 1b. This means that the overall structure of the F protein varies greatly among the different genotypes, which may have important consequences in relation to the functionality of the protein in different genotypes and subtypes.
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DISCUSSION |
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Strikingly, we found very high dn/ds ratios for all pairwise comparisons across all HCV genotypes for the F protein (i.e. dn/ds>1; see Table 2). One possible explanation for the F protein having such a large dn/ds ratio is that its non-synonymous variation is simply the result of variation in the overlapping core gene, which is in a different reading frame. Nevertheless, these results showed a different pattern of amino acid substitutions for the F protein than for the the core protein and other regions of the HCV genome.
We found a high degree of genetic variability and amino acid substitution rates along the F protein (Fig. 1). This is in contrast to the results commonly found in other viral systems, such as human immunodeficiency virus (Zanotto et al., 1999
) and hepatitis A virus (Costa-Mattioli et al., 2003
), and even with other HCV proteins, such as the core protein (not shown). This suggests that deterministic forces are not acting to conserve a particular domain or region.
The results of this work are in agreement with previous reports indicating that the F protein displays no clear sequence homologies to other proteins of known function, except that it is highly basic (Xu et al., 2001). Interestingly, the F protein does not appear to be essential for viral RNA replication, as its absence did not abolish the replication of an HCV RNA replicon in Huh7 hepatoma cells (Lohmann et al., 1999
; Blight et al., 2000
).
HCV chimeras have been constructed and shown to be infectious (Yagani et al., 1998) and the HCV F protein has been expressed (Roussel et al., 2003
). Taking this into account, specific experiments can be designed to determine whether HCV F proteins from different HCV genotypes show differences in specific functions, such as morphogenesis, replication and ligand interactions. These will provide a definitive picture of the role of the F protein in the biology of HCV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 August 2004;
accepted 27 September 2004.
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