Moloudi, Zahra; (2001) Autocatalytic processing of mutant SIVmac251-32H proteinase polyproteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The retroviruses HIV and SIV are structurally very similar, hence, SIV can be used as a model for HIV. There is much interest in developing treatment strategies for HIV, which is now responsible for more than 3.1 million deaths worldwide per year (UNAIDS & WHO, 2001). Many of the current treatment regimens are effective but inappropriate for use in the Third World. In this thesis, SIV was used as a model to investigate the effects of point mutations within the proteinase gene on autocatalytic cleavage, and to investigate the effect of cleavage site mutations on the autocatalytic cleavage mechanism. Initially, a purification protocol was developed for the wildtype SIV proteinases and its mutants. Purified enzymes were used to determine preliminary effects of these mutations on the activity of the proteinases. Wildtype, and SIV proteinase mutants were expressed in E.coli and purified from inclusion bodies yielding complete cleaved products. Differences were observed in the case of some mutants such as one termed G48V "extended proteinase" in which incomplete processing of the proteinase was seen. This raised the possibility that the incomplete processed proteinase could be used for therapeutic purposes such as vaccine therapy. The degree of in vitro processing was analysed by molecular weight de determination, and the compositions of these enzymes and their activities were also determined Purified 11 kDa proteinase was isolated from the wildtype, G48V "72", and L90M. Larger partly processed forms from partially purified proteinase were isolated from mutants, G48V "4833", and G48V "extended proteinase". SDS-PAGE analysis and Western blotting were used to identify the different forms of the proteinase. The 11 and 15 kDa proteins of the G48V "4833" clone were separated using gel filtration chromatography. The activities of the separated peptides were investigated. The N-terminus of these two proteins was shown to be identical by protein sequencing. Interaction between the two peptides affected stability and activities. The novel mutant G48V "extended proteinase" with a longer C-terminus was derived from the G48V "4833" gene sequence which was further manipulated by frame shifting at a convenient restriction site. Lower activities and differences between the predicted and observed proteinase size are described. Mutation in the N-terminal region of the proteinase may also slow down or lead to the incomplete autoprocessing. Mutants with variant proteinase activity may prove to be important for the development of future therapeutic strategies and further work is indicated.

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