October 2007

Document Type


Degree Name



Dept. of Molecular Microbiology and Immunology


Oregon Health & Science University


The work presented herein aims to characterize molecular interactions of importance to the assembly and maturation of human immunodeficiency virus type 1 (HIV-1). Production of infectious virus particles depends on proper protein-protein interactions, protein-membrane interactions, and protein-nucleic acid interactions. PrGag and PrGag-Pol proteins have to be targeted to functional assembly sites within the producer cell, interact in a specific fashion, bud off from the cells, and undergo a concerted mechanism of proteolytic processing in order to become infectious. Extensive X-ray and NMR studies have elucidated details about the structures of the HIV-1 capsid (CA) protein. The predominantly α-helical N-terminal domains (NTDs) have been modeled to form hexamer rings in the mature cores of virions, whereas the C-terminal domains (CTDs) are thought to engage in dimerization. A role for a histidine switch model involving one or more of the highly conserved histidine residues in the CA NTD had been proposed. We chose to examine substitutions at histidine residue 84 (H84), which has been modeled at the outside of hexamer rings, and H87, which is less well conserved and lies in the cyclophilin A (CypA) binding loop. Mutations at H84 resulted in poorly infectious virus with aberrant cores and low reverse transcriptase activities in cores, whereas mutations at H87 yielded virus with only a slight reduction in infectivity. Our results suggest that HIV-1 CA residue 84 may be involved in stabilizing CA monomer tertiary structure and contributes to an arrangement which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures. Membrane targeting of PrGag and incorporation of HIV-1 Env have been attributed to the matrix (MA) domain of HIV-1 PrGag. To evaluate the specific requirements for the MA membrane-binding domain (MBD) in HIV-1 assembly and replication, we replaced MA with alternative MBDs. We chose the pleckstrin homology (PH) domains from AKT protein kinase and phospholipase C δ1, as well as the cysteinerich binding domain of phosphokinase C γ. Our results demonstrated that alternative MBDs could promote VLP assembly and release, but the viruses were not infectious. Notably, PrGag processing was reduced, while cleavage of GagPol precursors resulted in the accumulation of Pol-derived intermediates within virions. Our results indicate that the HIV-1 assembly machinery can accommodate considerable variations in MA with regard to its means of membrane association, but that alternative MBDs can interfere with the structural rearrangement of virus cores during maturation.




School of Medicine



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