Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France1
Author for correspondence: Simon Wain-Hobson. Fax +33 1 45 68 88 74. e-mail simon{at}pasteur.fr
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Abstract |
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To see if GA hypermutation can be sustained over 10 kb of minus-strand DNA synthesis, this clone (Borman et al., 1995
) has been completely sequenced. All regions of the provirus were hypermutated including the two LTRs. Given that a typical HIV-1 base composition is approximately 36% A, 23% G, 19% C and 22% T and that of the hypermutated Vau genome is approximately 43% A, 16% G, 19% C and 22% T, this would suggest that approximately 31% of Gs were substituted. However, given the paucity of HIV-1 O sequences, as well as their divergence, the extent of hypermutation was difficult to ascertain. Accordingly a complete Vau sequence was assembled by sequencing four fragments amplified from the same Hirt DNA preparation from which the hypermutated provirus was cloned. Primers were designed from what appeared to be non-hypermutated regions. Upon cloning in a TOPO TA cloning vector, inserts were sequenced. Both strands were covered and all differences with respect to the two full-length published sequences, MVP5180 (Gürtler et al., 1994
) and ANT70 (Vanden Haesevelde et al., 1994
) were checked on the fluorograms. Accession numbers for the two Vau sequences are AF407418 and AF407419. Alternatively they can be found at ftp.pasteur.fr/pub/retromol/Vau.
The extent of GA hypermutation is shown in Fig. 1
in the form of differences with respect to the reference Vau sequence (Fig. 1
). A total of 680 out of 2189 Gs were substituted, or 31%, while the ratio of G
A/A
G transitions was 680/13. Typically, substitutions occurred mainly in GpG (60%) and to a lesser extent in GpA (34%) dinucleotides to the detriment of GpT (3%) and GpC (3%) (Borman et al., 1995
; Fitzgibbon et al., 1993
; Vartanian et al., 1991
, 1994
). Many runs of G were substituted reflecting once again the ability of the RT to elongate beyond mismatches, even runs of three to four mismatches.
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Not surprisingly, within gp120 Env variation is particularly evident. The V4 hypervariable region of all published HIV-1 O Envs show an additional disulphide bridge (Fig. 2). This is intriguing given that a similar situation pertains to a fraction of HIV-1 A/E clade viruses circulating in Thailand (Fig. 2
), although its precise location is slightly different (McCutchan et al., 1996
). It represents a case of parallel evolution, for this is the only feature in common with the A/E viruses. This extra disulphide bridge is absent in N and SIVcpz strains. Unfortunately this region of the Env structure was insufficiently ordered in the crystal structure to permit analysis.
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References |
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Received 9 October 2001;
accepted 26 November 2001.