1 Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France
2 Unité de Recherche Prévention et Thérapie moléculaires des Maladies humaines, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France
Correspondence
Jean-Pierre Vartanian
jpvart{at}pasteur.fr
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ABSTRACT |
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MAIN TEXT |
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In the singular context of a HIVvif virus, only APOBEC3F and -3G appear to be packaged into the virion (Harris et al., 2003
; Bishop et al., 2004
; Liddament et al., 2004
; Wiegand et al., 2004
; Zheng et al., 2004
). It is of note that APOBEC3F and -3G are packaged during budding from the donor cell and do not enter the replication complex of an incoming virion. Consequently, as soon as minus-strand viral cDNA is synthesized in the next round of infection, the numerous multiple C residues are deaminated, yielding U. Following plus-strand DNA synthesis, the U residues are copied into A, giving rise to so-called G
A hypermutants, by reference to the viral plus strand (Pathak & Temin, 1990
; Vartanian et al., 1991
).
As GA hypermutants are associated with a lethal phenotype, the absence of vif, their detection in a natural setting is, not surprisingly, highly variable and their frequency is often low. However, as G
A hypermutants frequently exhibit 2060 % of G residues substituted by A, their base composition is shifted considerably from that of the parental sequence. As AT-rich DNA melts at a lower temperature than DNA with a higher GC content, this suggested a means to differentially amplify AT-rich hypermutants, simply by modulating the PCR denaturation temperature. The converse is well-known: when working with GC-rich genes or genomes, PCR denaturation temperatures are usually increased to around 95 °C and occasionally higher (Smith et al., 1996
). Hence, it should be possible to find a denaturation temperature that allows amplification of AT-rich variants and not the parental sequence.
Low denaturation temperatures have rarely been exploited to differentially amplify AT-rich DNA. One report described ligation-mediated PCR performed at low denaturation temperatures (notably 8088 °C), to selectively amplify AT-rich segments within a bacterial genome (Masny & Pucienniczak, 2003
).
From previous work on HIV GA hypermutation, we had a large collection of molecular clones, corresponding to the V1V2 region of the HIV-1 envelope gene, that differed uniquely in the number of G
A transitions. A smaller region within this fragment was amplified by using Taq polymerase and degenerate primers that were derivatives of the standard SK122/SK123 pair (Goodenow et al., 1989
), to result in better amplification of hypermutated genomes. Their sequences were: SK122intD, 5'-AAARCCTAAARCCATRTRTA; SK123intD, 5'-TAATGTATGGGAATTGGYTYAA. When the PCR denaturation temperature was lowered to 83 °C (the reaction profile was 5 min at 83 °C, 25 cycles of 1 min at 83 °C, 30 s at 45 °C and 30 s at 72 °C, followed by 10 min at 72 °C), it was possible to uniquely amplify clones harbouring at least three mutations, whilst not amplifying the parental sequence (no mutations; Fig. 1a, b
).
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It is apparent from Fig. 1(b) that product recovery correlated with the extent of hypermutation. To explore more carefully the relationship between denaturation temperature and the number of G
A transitions per clone, another series of G
A-hypermutated reference clones spanning another locus within the V1V2 region was analysed by using Taq polymerase and a different pair of primers, RT3 and RT4 (Martinez et al., 1994
). Lowering the denaturing temperature by 1 °C progressively amplified more extensively hypermutated sequences (Fig. 1d
). Given the exquisite relationship between denaturation temperature and AT content of a sequence, the success of amplification may also depend on the calibration of the PCR machine and perhaps upkeep and make. Accordingly, all PCRs were performed on the same machine. These findings show that the selective amplification of G
A hypermutants is indeed generally related to the melting temperature of the target DNA. We refer to this method as differential DNA denaturation PCR, or 3D-PCR.
Fig. 1 also shows nested PCR material (293T/PBMC) corresponding to the same V1V2 region amplified from peripheral blood mononuclear cells (PBMCs) that had been infected with a
vif derivative of HIV-1 pNL4.3 following transfection of 293T cells. The denaturation temperature was 83 °C. The fact that this material represented differentially amplified G
A hypermutants was indicated when the 3D-PCR products were electrophoresed in a gel containing HA-yellow (Fig. 1c
, 293T/PBMC). When the 3D-PCR products were cloned and sequenced, the vast majority of sequences were extensively hypermutated, harbouring between three and 18 G
A transitions compared with the reference sequence (Fig. 2
). Of the 18 sites bearing G
A transitions, 15 were in the context GpA and three were in the context GpG. Cloning and sequencing of PCR material amplified at 95 °C identified only wild-type DNA (not shown). The surprise here is that the HIV-1
vif virus stock was made by using the 293T cell line, which is widely used as not only can it be transfected easily, but also it is considered not to express APOBEC3 molecules. From what is known of the mechanism of G
A hypermutation, the simplest explanation is that the 293T cell line had become clonally heterogeneous, so that APOBEC3F [preference for 5'TpC dinucleotide, GpA on viral plus strand (Harris et al., 2003
; Liddament et al., 2004
; Wiegand et al., 2004
; Zheng et al., 2004
)] as opposed to APOBEC3G [(5'CpC preference, or GpG on plus strand (Harris et al., 2003
; Lecossier et al., 2003
; Suspène et al., 2004
)] was being expressed in a subset of cells. Presumably 3D-PCR was picking up DNA from viruses produced by this subset.
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Among the ten samples, only one yielded a strong signal by 3D-PCR, with the following reaction profile: 5 min at 90 °C, 25 cycles of 1 min at 90 °C, 30 s at 45 °C and 30 s at 72 °C, followed by 10 min at 72 °C. When cloned and sequenced, a series of AT-enriched sequences was obtained, with substitutions mapping particularly to VP1 residues 560728 in the alignment of enteroviral polyproteins (www.iah.bbsrc.ac.uk/virus/picornaviridae/SequenceDatabase/alignments/entero_pep.txt). The sequences carried between one and six substitutions per segment. Of the 34 distinct substitutions, 28 were non-synonymous (including two nonsense), which is typical of variation within a quasispecies that has not undergone purifying selection. All but one substitution yielded genomes that were enriched in A and T. Amplification, cloning and sequencing of PCR material obtained at 95 °C revealed 17 clones that harboured only two substitutions in the locus shown in Fig. 3 (data not shown). Hence, it can be concluded that 3D-PCR was indeed amplifying the AT-rich end of the poliovirus mutant spectrum. As only one sample could be amplified differentially, the AT-rich variants presumably represent an unusually broad mutant spectrum and have nothing to do with the post-vaccination syndrome.
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In the precise setting of HIV, 3D-PCR has shown that one cell line that is used widely to support the replication of vif genomes is probably clonally heterogeneous, meaning that there is a background G
A-hypermutated signal in any sample. The ability to discriminate AT-rich variants over background suggests that this technique might find a variety of applications to biological questions.
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ACKNOWLEDGEMENTS |
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Received 7 July 2004;
accepted 2 October 2004.