Post-incorporation degradation of RT has been reported previously. Wapling et al. [35] have reported that mutations of W401L and W401A in RT can inhibit RT dimerization, resulting Fig. 6A . A big side chain (blue) is found in naturally occurring residues but not in 271A. (C) Structural models of naturally existing 274I, 274V and 274L residues as well as the lethal 274A mutation of p51 subunit. The same region was highlighted above in the right panel of Fig. 6A . A big side chain (pink) is found in naturally occurring residues I, V and L, but not in 274A. doi:10.1371/journal.pone.0006108.g006 [36] . Another mutation, L289K in p66 subunit, has also been reported to be able to abrogate dimerization [37] . Another group, when they tried to generate infectious molecular clones of Simian Immunodeficiency Virus, found that glutamic acid replacement at position 287 affected the stability of RT [38] . The biologically active and stable form of HIV-1 RT is a heterodimer consisting of p66 and p51, while the immediate precursor of p66/p51 heterodimer is the p66 homodimer. Proteolytic removal of the RNase H domain in one of the p66 homodimer subunit by HIV-1 protease leads to the formation of stable heterodimer p66/p51 [39] . RT exists as an equilibrium mixture of monomers and dimers that include the p66/p51 heterodimer and the p66/p66 and p51/p51 homodimers, among which the heterodimer is the most stable and the p51/p51 homodimer is the most unstable [40] [41] [42] [43] . Plausibly, the degradation of RT as reported in previous studies may be ascribed to the inhibition of RT p66 dimerization by the mutations. The mechanism of RT degradation in the virions observed in this study, however, may be different. Our results showed that RT mutants were still undetectable in the viruses generated in the presence of a potent dimerization enhancer (Fig. 5A ). Our native gel analysis showed that the dimer form of RT mutant 271A was detected, although it was markedly reduced as compared to the wild type, while mutant 274A could not form dimer. Instead, higher order oligomers of RT were detected in both mutants (Fig. 5B ). It has been reported that higher order oligomerization may occur in HIV-1 RT [41, 44, 45] . Our results have also indicated that Y271A and I274A substitutions may change the conformation of RT, leading to oligomerization. The higher order oligomers formed in these 2 mutants, perhaps tetramer and octamer, may be associated with instability of RT mutants and their susceptibility to viral protease. Furthermore, according to the locations of residues 271 and 274 (Fig. 6A) , it is highly probable that alanine substitutions at 271 and/or 274 can affect the conformation of p51 thumb region and subsequently its interaction with RNase H domain of p66, resulting in the exposure of the 7-amino-acid p51-RNas H cleavage sequence in the p66 subunit [7, 46] and consequent proteolytic degradation by HIV-1 protease. The relative conservation of RT 271 and 274 residues and the loss of big side chains at these two sites in the mutants as shown in the structural models (Fig. 6B & 6C ) also support their important roles. We tried to demonstrate this in vitro, by treating wild type and mutant RTs with recombinant protease, but wild type RT, as well as mutant RTs, could not be digested in vitro (data not shown). This result is consistent with that reported by Abram and Praniak [36] . This could be explained by different proteolytic stability of RT in vitro and in virions.