Summary: | 博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 87 === The human immunodeficiency virus (HIV) is the etiological agent of human aquired immunodeficiency syndrome (AIDS). It belongs to the Retroviridae family. All retroviruses share a basic genomic organization, i. e., R-U5-gag-pol-env-U3-R, and the pol of HIV-1 and many other retroviruses sequentially encodes protease (PR), reverse transcriptase (RT), and integrase (IN) from the 5'' to 3'' direction. For exploring the significance of this gene arrangement, all six possible gene dispositions were examined. We found that the HIV-1 protease was active when PR remained in the leading position of the reorganized Pols (eg., TF-PR-IN-RT), but no proteolytic activity was detected when IN was translocated to the front of PR-RT (i.e., TF-IN-PR-RT) or when PR was placed between RT and IN (i.e., TF-RT-PR-IN), even though the N-terminal junction of PR was cleavable. The TF/RT junction could be processed when PR was fused with the C-terminal end of RT-IN (i.e., TF-RT-IN-PR); no processing, however, was observed at the natural cleavage site of RT/IN, which was indeed cleavable by trans addition of a mature protease. The processing of TF-IN-RT-PR was also abolished. Lacking appropriate cleavage site, rather than lacking a functional protease, in this construct appeared to confer this observation. Based on the above findings, the C-terminus-processed PR precursor and mature PR may differ in their proteolytic activities. To test this notion, we generated two pairs of constructs, i.e., tf-pr-rt and tf-rt-pr, and tf-pr-in and tf-in-pr, to mimic mature PR or immature PR with only one end matured. Their products were further examined for trans activity on digestion of Gag and TF-RT substrates. The results showed that only the N-terminus-matured PR possesses a trans cleavage ability and this activity does not need a matured C-terminus. To exclude that the above observations are due to particular constructs, we generated an N-terminus-extended PR by inserting an extra Phe between TF and PR. This tf*pr construct indeed had a immatured N-terminus and expressed undetectable to a low level of trans cleavage activity. Additional constructs of tf*pr-rt and tf-pr*rt gave similar results all suggesting the notion that the N-terminus processing of PR is needed for activation of its trans activity. On studying a single chain-tethered protease dimer, we observed that a direct-linked PR dimer possesses a trans-processing activity. It may explain that the evolved HIV genome contains a single copy of PR. If HIV-1 contained two PR domains, PR would initiate the processing of polyprotein precursor within cytoplasm in the early stage of viral replication and might result in no infectious viral particle formation. Furthermore, interposition of a Phe at the P2'' position of the PR/PR junction did not affect its PR activity at all. However, a Phe was interposed at the P2'' position of the TF/PR junction, no trans activity of PR was observed. We also examined the effect of other amino acid residues interposed at the P2'' site of the tethered PR dimer. The results show that amino acid residues per se were more important than the length of amino acid residues added to the dimer junction. All together, our results provide imformation about how PR functions and why it is organized in a special manner in HIV-1 Pol polyprotein.
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