1 Unité de Rétrovirologie Moléculaire, Institut Pasteur, F-75724 Paris cedex 15, France
2 Department of Virology, University of the Saarland, D-66421 Homburg, Germany
Correspondence
Simon Wain-Hobson
simon{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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The first estimation of a retrovirus recombination rate, i.e. the number of RNA crossovers in a single round of replication, was made for spleen necrosis virus (SNV), an oncogenic avian retrovirus. The rate was 0·2 crossovers per genome per round (Hu & Temin, 1990
; Zhang & Temin, 1993
). In contrast, the recombination rate for the human immunodeficiency virus type 1 (HIV-1) lentivirus is of the order of about three per genome per round (Jetzt et al., 2000
; Yu et al., 1998
), more than 10-fold the rate for SNV. Importantly, for both viruses these recombination rates are 4- to 10-fold greater than the overall point mutation rates, which are
0·05 per genome per round for SNV (Pathak & Temin, 1990
) and
0·25 per genome per round for HIV-1 (Mansky & Temin, 1995
). In other words, by the time a mutation is made, template switching has occurred between 4 and 10 times.
Recombination is found at all levels of HIV genetics. Some HIV-1 M strains in global circulation are clearly composites of viruses from two to three clades (Carr et al., 1998; Hoelscher et al., 2001
; McCutchan, 2000
), while a few recombinants between HIV-1 M and O have been described (Peeters et al., 1999
; Takehisa et al., 1999
). Viral segments amplified from isolates or patient material have revealed numerous examples of recombination (Cheynier et al., 2001
; Gratton et al., 2000
; Vartanian et al., 1991
). In an experimental setting, wild-type simian immunodeficiency virus (SIVmac) could be recovered from peripheral blood of macaques co-infected 15 days earlier by two viruses carrying deletions in either the vif or the nef genes (Wooley et al., 1997
).
A recent report showed that the majority of HIV-1-infected splenocytes from two individuals harboured between one and eight proviruses, the mean being three to four per cell (Jung et al., 2002). Depending on the sequence divergence among proviruses within a single cell, the impact of recombination may be undetectable or easily identifiable. For example, if the two genomic RNAs packaged in a retrovirus particle were identical, it would be impossible to identify a recombinant. However, if the two genomes were substantially different, then recombination could be readily discerned. In the above study on single splenocytes, up to 2030 % amino acid sequence variation was noted within the first two hypervariable regions of Env (Jung et al., 2002
), including numerous recombinants. Multiple genetically divergent proviruses per cell means that the budding virions may contain RNA genomes derived from two different proviruses. Given m provirus copies per cell, there are m(m+1)/2 distinct ways to randomly assort m different genomic RNAs, among which will be m(m-1)/2 heterokaryons. For example, when m=4, there are 10 ways to reassort the genomes, of which six will be heterokaryons. Given a recombination rate of three crossovers per genome per cycle, virtually all heterokaryons will give rise to a unique mosaic structure.
Recombination can generate homoplasies that are the same character state in different genomes. However, homoplasies can also arise by independent RT misincorporation, which will be referred to as point mutation. Given that the HIV-1 recombination rate is 10-fold greater than the rate of point mutation, when in doubt it may be reasonable to rule in favour of recombination underlying any homoplasy as opposed to point mutation. Yet the overall rate of retrovirus point mutation is of course an average; the Km and Vmax of some substitutions, particularly transitions, will be greater than others and context dependent (Ricchetti & Buc, 1990). Thus, it might be argued that a few sites, constituting hot or warm spots, might rival recombination as an explanation for the existence of homoplasies. The question requiring an empirical answer is, do homoplasies in lentiviral genomes arise mainly from recombination or mutation?
If rampant recombination generates large numbers of homoplasies then what are the effects on branch lengths? Certainly, standard phylogenic methods ignore recombination. Computer simulations in which sequences were recombined show that branch lengths were overestimated (Schierup & Hein, 2000). Network analyses such as the SplitsTree program are more appropriate to describing homoplasies, recombination and sequence space (Bandelt & Dress, 1992
; Eigen & Nieselt-Struwe, 1990
; Huson, 1998
). Yet sequence space is enormous. For example, 100 variable sites typical of different molecular clones from the same isolate means connecting 4100 (
1060) points in sequence space. Inevitably, constraints have been introduced into the SplitsTree program. For example, no hypercube presentation of the sequence data is possible. Nonetheless, useful information can still be recovered (Cheynier et al., 2001
; Kils-Hütten et al., 2001
; Plikat et al., 1997
). Here, it is shown that for HIV and SIV, homoplasies are mainly the result of recombination. The implications for the analysis of virus evolution are discussed.
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METHODS |
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Consider a set of four closely related sequences (Fig. 1A). Sequences I and N differ by single substitutions (A2G and T6C) from the parental sequence H, while D encodes the same two substitutions. These sequences can be placed at the vertices of a square of unit length of one mutation (Fig. 1B
). D can be derived from either I or N by an independent transition, either A2G or T6C (Fig. 1C
). In this representation, the same substitution at the same site, yet in a different genome, i.e. homoplasy, shows up as parallel lines. The alternative solution is that sequence D arose by recombination of sequences I and N, somewhere between bases 2 and 6 (Fig. 1C
). It is clear that the results of the two different molecular events, recombination and point mutation, are indistinguishable by a posteriori sequence analyses.
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In contrast to site stripping, which may lead to erroneous topologies (Alber et al., 2001; Barbrook et al., 1998
; Holmes et al., 1999a
, b
; Smith et al., 1999
) (Fig. 2B
), eliminating the minimum number of sequences has the advantage of generating a correct topology for those sequences retained. This trial and error solution has been used in a few studies to analyse mutation rates and matrices among sets of closely related HIV or SIV sequences (Cheynier et al., 2001
; Kils-Hütten et al., 2001
; Plikat et al., 1997
).
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RESULTS |
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In a previous study of the evolution of the first two hypervariable regions (V1V2) of the SIV envelope protein, data from four animals at two time-points were reported (Pelletier et al., 1995). The sequences were under very weak selection pressure, a finding reiterated for an analysis of the equivalent regions in the HIV-1 Env protein (Kils-Hütten et al., 2001
). The interesting feature of the SIV data set was the fact that the infections were initiated by inoculation with DNA of an infectious molecular clone. Accordingly, all the sequences were derived from the same founder sequence and not a collection of variants derived during preparation of stock virus that would otherwise be used for inoculation.
For three animals, 20-188, 20-526 and 20-402, virtually all sequences could be incorporated individually in SplitsTrees with a fit of 100 (Fig. 3AC). There was evidence of some parallelograms but they were generally few. In contrast, for animal i-963, there was far greater evidence of network formation (Fig. 3D
). Homoplasies, i.e. the same substitution at the same site, among sequences from the four animals are highlighted in colour. There were 15 homoplasies among a total of 125 discrete substitutions, 14 shared between two animals and one (A68G) between three animals. However, this A68G transition was present only in sequences taken at 19 months post-infection, indicating that it is hardly a hot spot of mutation. Equally, a substitution connecting a taxon to the parental SIVmac239 in one tree was rarely connected to SIVmac239 in another tree, again indicating that they cannot be described as hot spots. Overall, the fraction of true homoplasies due to point mutations between animals represent a minority of total number of substitutions (excess substitutions=14x1+1x2=16; excess/total discrete substitutions=16/125,
13 %), indicating that this is probably also true of substitutions within an animal. Homoplasies between animals could be broken down into 10 non-synonymous substitutions, four synonymous substitutions and one transversion generating an in-phase stop codon. Such a distribution is close to that expected from weak purifying selection, as noted before (Pelletier et al., 1995
).
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Previous work has shown that the analogous region of the HIV-1 env gene is also diversifying under weak selection and shows extensive networks by SplitsTree analyses (Cheynier et al., 2001; Kils-Hütten et al., 2001
). Furthermore, the same region was studied for HIV-1 DNA from laser microdissected single cells (Jung et al., 2002
). When analysed by SplitsTree, the sequence sets from individual cells showed extensive networking (Fig. 5
A, B). Given that homoplasies in the SIV V1V2 locus arose mainly by recombination, it is reasonable to assume that these networks too arose mainly by recombination. As before, the minimum path lengths were shorter when recombination was factored in 16 and 9 % for the two examples in Fig. 5(A, B)
, respectively.
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When may mutation generate extensive networking? Monotonous GA hypermutation of retroviral genomes occurs during negative-strand DNA synthesis under conditions of an unbalanced dTTP and dCTP concentration (Martinez et al., 1994
; Vartanian et al., 1997
). Overall, G
A substitution frequencies of up to 0·3 per G residue are possible (Vartanian et al., 2002
). As an example, a small segment of a set of hypermutated HIV sequences generated in vitro is shown in Fig. 7
(A). SplitsTree analyses of the sequences generated extensive networks (Fig. 7B
). Thus, when the point mutation rate is very high,
104-fold greater than the normal rate of
0·25x10-5 per base per cycle, as is the case here, extensive networking due to point mutation is possible. In turn, this indicates that at normal mutation rates, recombination is the major mechanism underlying network formation.
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DISCUSSION |
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As can be seen when scoring the number of mutations, ignoring recombination inflates the minimum path length connecting sequences in any data set (Figs 1, 46). Among the examples shown there was an overestimation of between 10 and 45 %. This conclusion is derived empirically. It is intriguing that the upper value is close to the 50 % predicted from the simplest conceptual example shown in Fig. 1(C)
. Hence, from the temporal standpoint, intrapatient HIV lineages may be generally younger than might be otherwise anticipated.
When factoring in recombination, the minimum number of point mutations is simply the number of discrete substitutions across all variable sites in the data set. This obviously reduces statistical power when analysing mutations in terms of non-synonymous and synonymous substitutions (Zanotto et al., 1999). Furthermore, by ignoring both phylogeny and recombination, two-by-two sequence comparisons used to compute ratios of non-synonymous to synonymous substitutions and thence infer positive selection are equally erroneous (Evans et al., 1999
; Price et al., 1997
). The statistical analysis of codon polymorphisms has received considerable attention of late (Nielsen & Yang, 1998
; Yamaguchi-Kabata & Gojobori, 2000
; Yang et al., 2000
). In fact, sequence data in Fig. 2
corresponded to nef codons that were identified in a PAML analysis as being under statistically significant positive selection (Nielsen & Yang, 1998
; Yang et al., 2000
; data not shown). The minimum path length connecting all 16 taxa in Fig. 2(D)
requires 17 substitutions, among which there are seven homoplasies (Fig. 8
A). Allowing for recombination, the minimum of substitutions necessary is 11 (Fig. 8B
), the number of discrete substitutions in the 16 taxa data set (Fig. 2A
). In short, each substitution could have occurred just once and spread by subsequent recombination. It would seem that inferring positive selection from the statistical analysis of polymorphic codons may be invalid if recombination is taken into account.
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This situation where the recombination rate exceeds the mutation rate is not without precedent. For numerous bacteria, including Neisseria species, Streptococcus pneumoniae and Staphylococcus aureus, evolutionary change at neutral loci is more likely to occur by recombination than by point mutation (Feil et al., 1999, 2000
, 2001
). Splits decomposition analyses have revealed extensive networks (Alber et al., 2001
; Holmes et al., 1999a
; Smith et al., 1999
). Estimates of the recombination to substitution rates may be as high as 100 : 1 (Feil & Spratt, 2001
), although precise recombination rates are lacking. Yet, as the point mutation rates for RNA viruses and retroviruses are far higher than those of bacteria, the sheer profusion and tempo by which retrovirus recombinants arise distinguish them from their bacterial counterparts.
In conclusion, HIV and SIV sequence polymorphisms in a data set are strongly influenced by recombination. When recombination is catered for by the SplitsTrees program, the minimum number of substitutions in a data set necessary to explain sequence complexity is the number of unique mutations. Accordingly, a sequence set is younger than would have been thought previously.
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ACKNOWLEDGEMENTS |
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Received 10 October 2002;
accepted 13 December 2002.