Abstract
Human immunodeficiency virus 1 (HIV-1) is one of the deadliest retrovirus known to mankind due to its ability to target our immune cells including, CD4⁺ T cells, CD8⁺ T cell, macrophages, monocytes, and dendritic cells, which leads to an acquired immune deficiency syndrome (AIDS). Throughout evolution, HIV-1 has developed efficient mechanisms in manipulating the host immune system through the help of its accessory proteins that ensure a permissive environment to its replication and spread. Among those is a viral protein called Nef, which is a master in hijacking cellular membrane trafficking pathways to downregulate important immune receptors, including MHC-I, CD4, etc. Adding to that, only recently, antiviral transmembrane proteins in the host cell called SERINC3 (S3) and SERINC5 (S5) were discovered to decrease HIV-1 infection by blocking the newly budding virus from entering and infecting other cells. It has been shown that Nef also downregulates S3 and S5 as well by initiating endocytosis and degradation through the help of clathrin adaptor protein AP2, increasing viral infection. Mechanistic details of some downregulation events started to emerge only very recently. However, much remains to be learned and revealed about Nef. In pursuing the mechanism of S3 downregulation in Chapter II of this thesis, the binding of each intracellular loops of S3 with Nef was tested in vitro using Size Exclusion Chromatography (SEC) along with SDS-PAGE. Out of all the intercellular loops, the N-terminal loop of S3 gave the strongest affinity to Nef, giving an insight to the molecular mechanism behind the downregulation of S3 by Nef viral protein. In addition to S3, the downregulation of CD8 by Nef-mediated AP2-dependent endocytosis was also examined as discussed in Chapter III. In vitro, the binding test showed a strong affinity between Nef and CD8b using SEC. In addition, a pull-down assay showed an enhancement in binding between Nef and AP2 in the presence of CD8b cytoplasmic domain, suggesting that the assembled complex recapitulates the binding invivo. Furthermore, neurological association of HIV-1, Nef had also been reported as discussed in Chapter IV. HIV-1 is able to cross the Blood Brain Barrier, causing numerous neurological symptoms all together grouped in the HIV-Associated Neurocognitive Disorders. Some of these disorders are caused by a disruption in the myelin sheets. Previous reports had indicated an association between Nef viral protein and neuron host proteins including proteolipid protein 1 (PLP1), and Glycoprotein M6-B (GPM6B). These neuron host proteins serve a critical role in the biosynthesis of myelin sheets. Binding test using SEC had showed a strong affinity between Nef viral protein and host neural proteins (PLP-1, and GPM6B). This may explain some of the molecular mechanism behind the neurological disorders found in HIV-1 patients. Understanding the underlying molecular mechanisms for the downregulation of host cellular proteins by Nef, may enable us to explore antiviral drugs to inhibit HIV-1 Nef.