Table 3

Mutations in HIV RT

AnalogNo. of clonesMutations/nucleotides%No. of mutations
G→AA→GC→TT→CT→AA→CA→TG→TT→GC→AG→C
Controls and 5-OH-dC-induced mutations
Control818/4,3060.4227250002000
5-OH-dC2872/15,0600.483918441111201
Controls and lethal and breakthrough mutations
Control8599/47,8550.21343015190010000
5-OH-dC (lethal)95196/53,4850.37942633271732021
5-OH-dC (breakthrough)2324/12,9490.18413030310100
  • DNA sequence analysis of a segment of the HIV-1LAI RT gene spanning nucleotides 2193–2867. The segment of the HIV RT gene was amplified from proviral DNA obtained from frozen cells after the number of serial transfers indicated below. In the first two lines, the proviral DNA was amplified from control and 5-OH-dC-treated cells obtained at the 16th passage (see Fig. 2). In the bottom three lines, proviral DNA was obtained at passages indicated in Fig. 3. The method for amplification and cloning is similar to that described above, except that proviral DNA was purified by Isoquick Kit (Orca Research, Bothell, WA) and amplification was performed only with inner primers. The amplified DNA was ligated into the PCR II TOPO TA cloning vector (Invitrogen). The arrows in Fig. 3 indicate the cultures sampled and combined prior to obtaining viral DNA for amplification, cloning, and sequencing.