Mutations Y271A and I274A did not affect Pr160 gag-pol expression and transportation We next investigated whether the loss of RT in viral particles of the mutants was attributed to altered expression and transportation of the precursor protein, polyprotein Pr160 gag-pol , during virus assembly. Pr160 gag-pol in lysates of cells transfected with the wild type and mutants was detected by Western blotting. As shown in Fig. 3A , similar levels of Pr160 gag-pol , Gag protein (Pr55 Gag ) and capsid protein p24 (CA p24) were found in cells transfected with the wild type or mutant constructs, indicating that the expression and stability of RT precursor protein were not affected by the mutations. Immunofluorescence staining further demonstrated a normal subcellular localization of mutant Gag-Pol polyproteins as compared to that of the wild type, suggesting that the transportation of Gag-Pol was unlikely to be affected by alanine substitutions (Fig. 3B) .