Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue de Dr Roux, F-75724 Paris cedex 15, France1
Abteilung Virologie, Universität des Saarlandes, Institut für Medizinische Mikrobiologie und Hygiene, D-66421 Homburg, Germany2
Author for correspondence: Simon Wain-Hobson. Fax +33 1 4568 8874. e-mail simon{at}pasteur.fr
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Abstract |
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Introduction |
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Recombination can also result in the formation of insertions and deletions (indels). These are nowhere more in evidence than in the segments corresponding to the hypervariable regions of the envelope protein. As opposed to point mutations, the rate of fixation of indels is not known. Certainly, indels are observed within a matter of months of infection (Burns & Desrosiers, 1991 ). However, there is no more precise estimation than this and there is certainly no study ranking the fixation rate of indels with respect to transitions and transversions. Of course, indels are invariably stripped from multiple alignments. In the case of the hypervariable regions of the primate immunodeficiency viruses, where indels are common, gap-stripping restricts the understanding of their evolution.
Phylogenetic reconstruction is needed in the counting of mutations; otherwise, there is a tendency to inflate their number (Pelletier et al., 1995 ; Plikat et al., 1997
; Zanotto et al., 1999
). The SplitsTree program creates networks of sequences representing parallel mutations and allows sequences to be placed at nodes as well as tips of a phylogram (Huson, 1998
). This is particularly appropriate when analysing early quasispeciation by an RNA virus or retrovirus such as HIV or SIV (Dopazo et al., 1993
; Pelletier et al., 1995
; Plikat et al., 1997
).
In principle, indels can be coded and treated as single events as long as the degree of sequence diversification is not too great, a condition met in the first 23 years of an HIV or SIV infection. Although a few reports have attempted to code indels (Barriel, 1994 ), none has applied the technique to RNA viruses, retroviruses or HIV/SIV. Here, a short program has been written that allows indel coding within sets of sequences and which is adapted to the SplitsTree program. When applied to the first hypervariable region of the SIVmac251 env gene, it was found that the fixation rate of deletions rivalled that of transitions.
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Methods |
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Deletions were similarly coded by 0 or 1 added 3' of the sequence after the coding of insertions. 0 means the absence of the deletion while 1 scores the deletion. Obviously, gaps in sequences were considered to be the same event when the borders were identical. Two sequences bearing deletions of say 6 and 9 bp, with only one common border, were treated as two independent events rather than a common 6 bp deletion and an extra 3 bp deletion juxtaposed in one sequence. After the coding process was performed, each gap was replaced in each sequence by the consensus bases derived from the remaining sequences without gaps.
In cases where there was no consensus among the remaining sequences, bases were chosen according to the following nucleotide frequency rules: A=T>G>C, A=G>T>C, A=C>G>T, A=T=C>G, A=T=G>C, A=C=G>T or A=T=C=G; A was chosen. T=G>A>C, G=C>A>T or T=C=G>A; G was chosen. T=C>A>G; T was chosen.
These rules were adapted to work with HIV and SIVs and reflect the general base composition of the primate lentiviruses (A>G>T>C) (Wain-Hobson et al., 1985 ). This can be modified. The set of 698 sequences analysed corresponds to the region of SIVmac251 encoding the hypervariable V1 loop of the envelope protein (Cheynier et al., 1998
).
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Results |
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The combination of indel coding and scoring mutations over SplitsTrees was applied to an extensive set of 698 sequences derived from a SIVmac251-infected macaque (Cheynier et al., 1998 ). Sequences from 1521 weeks post-infection were analysed together (Fig. 2
); a total of 40 could be introduced into a network with a fit of 100%, i.e. without removal of any mutations. There was some evidence of networking, which may reflect homoplasies and/or recombination. Choosing the minimum path length connecting all sequences, 56 point substitutions and five deletions were mapped among 40 sequences. By 6164 weeks post-infection, there was extensive networking; the most parsimonious path length scored 35 substitutions and seven deletions among the 30 sequences (Fig. 3
). In both instances, the numbers of deletions were comparable to the number of transversions.
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Discussion |
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Overlaying nucleotide substitutions onto the SplitsTrees and comparing observed and expected non-synonymous and synonymous substitution showed scant evidence of selection within the hypervariable V1 region (data not shown). The accompanying comprehensive analysis using the SplitsTree approach of a large number of different loci and published datasets tends to show that the majority of sites are not under positive selection (Kils-Hütten et al., 2001 ). This is not to say that positive selection is not operative, just that, using the ratio of non-synonymous to synonymous substitutions in segments of several hundred bases, the signal is generally too weak.
The possibility of recombination contributing to sequence complexity is apparent (Fig. 4). Distinguishing between homoplasies and recombination is not easy. However, hot spots were not much in evidence in sequences corresponding to the hypervariable regions of SIV and HIV Env, suggesting that some of the sequences were indeed recombinants.
It appears that indels contribute considerably to the evolution of the hypervariable regions and are more frequent than transversions. Insertions in these regions are invariably duplications and involve N-linked glycosylation sites, almost all of which are occupied. The carbohydrate moieties are thought to mask the hypervariable regions from recognition by immunoglobulins (Kwong et al., 1998 ; Wyatt et al., 1998
). Other regions of the HIV genome generally do not support so many indels (Alizon et al., 1986
), so the indel fixation rates revealed here represent upper values. Whether the unusually high A content of this region, with its capacity for propeller-twisting hydrogen bonds in oligo(A):oligo(T) tracts (Burkhoff & Tullius, 1987
), is conducive to indel formation remains to be established.
In conclusion, indel coding allows the estimation of indel frequencies with respect to point mutations and, in so doing, recovers more information from a set of sequences. The method could be useful in other contexts, such as estimating the frequencies of bacterial variable tandem repeats, and could be adapted to encompass the appearance on large additions and deletions of blocks of DNA when comparing bacterial genomes (Ochman et al., 2000 ).
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Acknowledgments |
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References |
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Received 20 October 2000;
accepted 8 March 2001.