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Sequences within Pr160gag-pol Affecting the Selective Packaging of tRNALys3 into HIV-1 H. JAVANBAKHT*, A. KHORCHID, S. WISE, M. A. PARNIAK, M. A. WAINBERG, and L. KLEIMAN. Lady Davis Inst. for Med. Res. and McGill AIDS Ctr., Jewish Gen. Hosp., Montreal, Canada The selective packaging of the primer tRNALys3 into HIV-1 particles is dependent upon the viral incorporation of Pr160gag-pol protein. We have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNALys3, using a series of deletions, insertions, and point mutations. These mutations were placed in a protease-negative HIV-1 proviral DNA to prevent protease degradation of the mutant Gag-Pol protein. C-terminal deletions of Gag-Pol which removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not affect the selective incorporation of either the truncated Gag-Pol or the tRNALys3, implying these regions are not required for tRNALys3 binding. C terminal deletions which included the 24VQPI244 sequence at the junction of the thumb and palm subdomains of RT prevented selective tRNALys3 incorporation. This sequence has been shown to interact with the 3` terminus tRNALys3 in an in vitro interaction between mature RT and tRNALys3. The thumb region point mutations, K249E, K249Q, and R307E, which have also been reported to inhibit the in vitro interaction of mature RT with the anticodon loop of tRNALys3 , did not inhibit tRNALys3 packaging into the virus, which is supportive of other data indicating that the anticodon loop of tRNALys3 is not involved in interactions with Pr160gag-pol during tRNALys3 packaging. On the other hand, insertion mutations in the connection domain of RT in Gag-Pol did prevent selective packaging of tRNALys3 without inhibiting mutant Gag-Pol incorporation. Since deletion experiments show these sequences are not required for tRNALys3 packaging, the insertion mutations must create an altered Gag-Pol configuration with decreased ability to bind tRNALys3. Key Words: Hiv-1, packaging, tRNALys3 |
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© 7th
Conference on Retroviruses and Opportunistic Infections, |