Figure 2

Sequence analysis of MMTV(C3H)hp1 from the second round infected Hs578T cells. Genomic DNA isolated from Hs578T cells 2 weeks after infection with MMTV(C3H)hp1 or cDNA synthesised from RNA extracted from filtered producer cell (Mm5MT) culture medium was used for amplification and cloning of the entire MMTV genome, minus the gag gene. (A) The ratio between the number of non-synonymous mutations per non-synonymous site (dN) and synonymous mutations per synonymous site (dS) was calculated using the Tamura-Nei method implemented in the MEGA software package that estimates the number of non-synonymous and synonymous changes that have occurred at each codon throughout evolution [33]. In the right panel, the dN/dS ratio for subregions in the MMTV env analysed using a sliding window of 100 codons is shown. (B) Sequence alignment of amino acid residues 1 through 72 of MMTV SU of two virus clones [MMTV(C3H)] and twelve proviral clones [MMTV(C3H)hp1] were aligned. Mtv-1 (AF228550.1), Mtv-2, Mtv-8 (M22028.1), Mtv-17 (AF263910.1), MMTV(C3H-HeN) (AF228552.1), MMTV(C3H-HeJ) (AF2258551.1), MMTV(RIII) (AF071010.1) and MMTV(BR6) (M15122.1) sequences were also included in the alignment. The coordinates of the MMTV Env amino acid sequence are according to Zhang et al. [20]. Potential N-glycosylation sites (GS) are highlighted in light green and RBS in yellow. (C) Predicted 3D structure of the MMTV SU in space-filled form generated using Swiss-Model is shown. The RBS is indicated in yellow, the glycosylation site in green, basic residues in blue and acidic residues in red. In the right panel, the RBS is enlarged and the charge change caused by G to E substitution at position 42 is highlighted.