Human immunodeficiency virus (HIV) is a member of a subgroup of retroviruses that lead to HIV infection and ultimately result in acquired immunodeficiency syndrome (AIDS), one of the most fatal viral diseases in human history. There are two types of HIV: HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1, which was discovered first, is much more contagious and virulent than HIV-2. In addition, HIV-1 is the cause of the majority of HIV infections all over the world. HIV-2 is less severe due to its relatively low infectivity. HIV viruses infect pivotal cells of the human immune system to lead to the failure of patient’s immune system. Dormancy of HIV viruses is comparatively long and the average survival time after HIV infection is 10 years without treatment. So far, millions of people around the world are suffering from HIV infection while there is no good way to treat it completely. Therefore, it is extremely important to study the process of HIV assembly to better understand the mechanism of HIV infection.
HIV assembly is an extremely complicated multi-step process. It takes place at the plasma membrane and involves the polyprotein Gag, genomic viral RNAs and lipids phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). During the early stage of HIV assembly, Gag is all that is required to assemble into a spherical, membrane-enveloped immature virion with a diameter ranging from 100-150nm. The Gag has an approximate mass of 55kDa and consists of six structural domains. From N-terminus to C-terminus, they are matrix (MA), capsid (CA), spacer peptides 1 (SP1), nucleocapsid (NC), spacer peptides 2 (SP2), and p6, respectively. The three major functional domains are MA, CA and NC. MA domain prefers to interact with PI(4,5)P2 on the lipid plasma membrane during HIV assembly. CA-CA interactions form hexagonal lattices with an approximate spacing of 8nm in the immature virion. NC domain has a high affinity to bind with the RNA packaging signal (Ψ) located in the 5’-end of a dimeric viral RNA that subsequently forms the conical shape virus core.
In this dissertation Atomic Force Microscopy (AFM) was utilized to study the morphology of Gag, ΨRNA, lipid PI(4,5)P2 as well as their different mixed complexes. They were imaged on either positively charged or negatively charged micas depending on the net charges carried by the respective materials. After achieving high quality AFM images in a liquid environment, programming scripts coded in MATLAB and Mathematica were used to analyze the data statistically. The sizes of the corresponding structures and the statistical distributions of Gag among monomer, dimer and tetramer complexes before and after mixing with either ΨRNA or PI(4,5)P2, or both were found. Then the binding principle of HIV Gag with ΨRNA and PI(4,5)P2 was investigated.