Sequences within Pr160gag-pol affecting the selective packaging of primer tRNALys3 into HIV-1

Abstract

The selective packaging of the primer tRNALys3 into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNALys3 binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNALys3. Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNALys3, indicating that these regions are not required for tRNALys3 binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNALys3 packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNALys3. While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNALys3, we find that these mutations do not inhibit tRNALys3 packaging into the virus, which supports other work indicating that the anticodon loop of tRNALys3 is not involved in interactions with Pr160gag-pol during tRNALys3 packaging.