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Abstract

Human Immunodeficiency Virus-1 (HIV-1) assembles and buds as non-infectious particles at the plasma membrane of host cells. During assembly, it forms an irregular hexameric ordered lattice consisting of the structural polyproteins Gag and GagProPol. To facilitate infectivity, the intrinsic protease (PR) of HIV-1 drives the maturation by processing these polyproteins at 5 and 9 cleavage sites (CS), respectively, after self-cleavage. This proteolytic part is tightly regulated temporally and sequentially, to ensure a correct morphological rearrangement by a specific release of subdomains inside the viral particle. Gag comprises the matrix (MA), capsid (CA), and nucleocapsid protein (NC) as well as two spacer peptides (SP1 and SP2) and the C-terminal p6-domain. After maturation, CA encapsulates a condensed complex of NC and the RNA genome copies as the conical core. Already subtle dynamical or structural changes can prevent a successful maturation and therefore impair viral infectivity. The concomitant start of maturation and assembly/budding processes so far prevented a precise time-course analysis of maturation in connection with structural and cofactor interactions, and the determination of pH during maturation. Previous proteolytic cleavage studies with in vitro translated Gag and viral particles already led to the categorization of the CSs regarding their processing rates: rapid (SP1-NC), intermediate (MA-CA, SP2-p6), and slow (CA-SP1, NC-SP2). However, these dynamics differ from the processing of synthetic CS peptides. That is why this work aimed to analyze the impact of Gag assembly, which is usually induced by the binding of nucleic acid, and other factors as specific mutations upon the dynamics of maturation. In previous studies, only the final products of maturation were analyzed in viral particles regarding morphology and processing results. Other time-course analyses excluded the verification of the Gag multimerization or the influence of nucleic acid as present in virus-producing cells. Thus, I wanted to compare the processing of Gag in an assembled structure or non-assembled state and additionally introduced specific cleavage site mutants and maturation altering compounds into my system. In order to tackle these open questions for Gag processing dynamics, an in vitro based processing approach for the analyses of proteolytic maturation was chosen, including non-assembled Gag and in vitro assembled ΔMACANCSP2, a truncated Gag variant. Therefore, I produced recombinant Gag with a C-terminal His-tag (Gag-His) in E. coli and optimized the protocol to yield high purity and no nucleic acid contamination, to avoid preliminary assembly. A given protocol to assemble ΔMACANCSP2 was optimized, which increased assembly efficiency and stability to endure the inconvenient conditions of the following processing experiment. Additionally, this newly created protocol could achieve assembly of ΔMACANCSP2 in the absence of any nucleic acid into curved filaments instead of spherical particles. As these filamentous structures are a novelty, further structural analysis of them could give more insight into the assembling properties of Gag in the future. Gag-His featured, independent of its intrinsic homodimerization, an altered order of processing in contrast to the assembled ΔMACANCSP2. The initial cleavage occurred at MA-CA and CA-SP1, followed by SP1-NC and SP2-p6, and at last, NC-SP2. In comparison, the processing of assembled ΔMACANCSP2 reproduced the same processing order shown in the literature, which was only marginally affected by the application of longer NA than 68 nt. The processing of assembled protein was finished up to six times faster than for the non-assembled, while the absence of or very shot (5 nucleotides) NA during the processing of assembled ΔMACANCSP2 caused a mixture of both results. While MA-CA and CA-SP1 are processed like assembled ΔMACANCSP2 with nucleic acid, SP1-NC and NC-SP2 are processed significantly slower than in the case of non-assembled Gag-His. These results suggest that the cleavage events of non-assembled Gag is dependent on the amino acid sequence of the CSs, and assembly causes for MA-CA and CA-SP1 a maturation restriction. Changing the pH of the processing procedure had a severe impact on the processing of CA-SP1 and NC-SP2 in an assembled or non-assembled protein. While the processing was fastest at pH 6.0, the optimum for PR activity, the processing was strongly reduced at pH 6.5 and even more at pH 7.0. The remaining three CSs were only marginally affected. Consequently, CA-SP1 and NC-SP2 might comprise pH-dependent structural domains or interactions, and a theoretical pH shift during maturation of viral particles could enable fast processing. The introduction of mutations known to inhibit proteolytic cleavage or a maturation inhibitor showed that the processing of each site, but CA-SP1, is independent of the cleavage of the other CSs. By inhibiting the processing at SP1-NC, the cleavage of CA-SP1 got delayed, which was observed in the presence and absence of nucleic acids. Interestingly, the inhibition of MA-CA cleavage led to the processing of a new cryptic CS, which was determined to be at the N-terminal region of CA. In summary of this work, assembly of Gag in the presence of nucleic acid accelerates maturation notably, whereas the single cleavage events are independent of each other and only temporally ordered. While assembly delays the processing at MA-CA and CA-SP1, the presence of nucleic acid is the actual key player to shorten the maturation, but it is not essential for Gag assembly.