Biological Sciences, School of
Date of this Version
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus and the causative agent of the acquired immune deficiency syndrome (AIDS) pandemic. The retrovirus replication cycle is divided into early infectious events, which involve the infection and integration of the viral DNA into target cell chromosomes; and late events, which involve the expression of viral genes and assembly of infectious virions. To complete the replication cycle, HIV-1 utilizes various cellular pathways.
We identified the Ubc9 E2 SUMO conjugating enzyme as a HIV-1 Gag interaction partner. When this interaction was disrupted in HIV-1 producer cells by Ubc9 siRNA, the virus that was released from the cells was approximately 10-fold less infectious than virus released from control cells. The decreased virion infectivity was found to be due to decreased levels of intracellular mature Env, which in turn decreased the amount of Env packaged into assembling virions. Surprisingly, the defect in mature Env levels in Ubc9 knockdown cell was dependent upon Gag expression. The mechanism of decreased mature Env in Ubc9 knockdown cells was examined and we found that gp120/gp41 appeared to be preferentially degraded at a post gp160 cleavage step, but before transport/association with the plasma membrane and lipid rafts. The intracellular gp120 levels were restored when cells were treated with lysosome inhibitors, which suggested that the decreased intracellular Env stability is due to increased lysosomal degradation; however, inhibiting Env degradation did not restore Env packaging into assembled virions. Since lysosomal degradation is linked with autophagy, we examined autophagy in Ubc9 knockdown cells and found that autophagy was increased approximately 3-fold compared to control cells. Autophagy inhibitors were unable to block autophagy, and we were unable to determine if increased lysosomal degradation of gp120 was occurring through an autophagy dependent or independent mechanism.
We have also examined the potential role of Ubc9 during the HIV-1 early infectious events. Decreased LTR driven gene expression was detected following the infection of Ubc9 knockdown cells with VSV-G pseudotyped HIV-1 virions. These results suggested that Ubc9 plays a functional role during the early events of the HIV-1 replication cycle at a post-entry step.
Adviser: Charles Wood
A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biological Sciences, Under the Supervision of Professor Charles Wood. Lincoln, Nebraska: September, 2011
Copyright (c) 2011 Christopher R. Bohl