Since HIV-1 RT is incorporated into the virion in the form of Pr160 gag-pol , which is transported to cell membrane where the virus is packaged, through interaction with Pr55 gag [27] [28] [29] [30] , we thus investigated whether the mutations would affect Pr160 gag-pol expression and transportation. Our results showed that Pr160 gagpol was properly expressed and transported in cells transfected with mutant constructs, Y271A and I271A (Fig. 3) , indicating that these mutations did not affect either the production of the precursor or the interaction between Pr160 gag-pol and Pr55 gag . It was further found that the Pr160 gag-pol was indeed incorporated into the virions of these two mutants, because except for RT, all other products of Pr160 gag-pol including p24, protease and integrase [31, 32] , could be detected in the viral particles of mutants Y271A and I271A (Fig. 4A) . Although it has been reported that the domain between residues 183 and 305 of RT is likely responsible for RT incorporation [33] , our study ruled out the involvement of RT was detected in wild type and mutant pseudoviruses generated in the presence and absence of EFV, the most potent dimerization enhancer, by Western blotting using mouse monoclonal anti-RT and anti-CA p24 antibodies, respectively. (B) Purified wild type and mutant RT subunit p66 were analyzed by native gel electrophoresis followed by Western blotting using mouse monoclonal anti-RT antibody in the presence or absence of b-mercaptoethanol (b-ME). Compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271A and 274A mutants formed higher order oligomers, suggestive of conformational change. doi:10.1371/journal.pone.0006108.g005 the turn (Gln 269 -Arg 277 ). Another report has suggested that two mutations in RT, L234D and W239A, led to premature cleavage of the Gag-Pol precursor and reduced levels of viral enzymes in the virions [34] . Our results also excluded that the mutations of Y271A and I271A affected the cleavage of the Gag-Pol precursor. By a viral protease inhibition assay using indinavir, we finally demonstrated that the RT mutants were degraded after incorporation of the precursor polyprotein into the virions and the degradation was associated with the activity of viral protease (Fig. 4C) . Our results indicated that the mutations in residues 271 and 274 would reduce the stability of RT inside the viral particles, rendering it susceptible to viral protease.