Shi, Binshan Theses Advised
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Item HIV-1 infection by cell-to-cell transmission induces human innate immune response.(Albany College of Pharmacy and Health Sciences Theses, 2023-07) Sanchez, Anthony '23HIV-1 can spread from infected cells to intact cells frequently through close cellular contact in human lymphoid tissues/organs. Compared to cell free virus infection, HIV-1 cell-to-cell transmission yields a significantly higher multiplicity of infection and requires both cellular membrane and skeleton changes, however the impact of HIV-1 cell-to-cell transmission to host innate immune response, particularly the IFN initiation by cGAS-STING pathways, has not been reported previously. In this study, it was found that HIV-1 GFP+ (∆env) virus in infected MT2 cells could transmit to uninfected MT2 cells and other immune cells predominantly by cell-to-cell transmission. The infection by cell-to-cell transmission of HIV-1 started to increase 12 hours and peaked at 36 hours after coculture of infected donor MT2 cells and uninfected recipient cells. Syncytia was observed in HIV-1 cell-to-cell transmitted MT2 cells by using fluorescence microscopy. In addition, the flow cytometry result indicated cell morphology and structure changes in MT2 cells after HIV-1 cell-to-cell transmission. Importantly, in HIV-1 GFP+ infected MT2 cells, phosphorylation of both STING and IRF3 proteins were detected at 36 hours post infection, which was virus infection dosage dependent. The nuclear translocation of phosphorylated IRF3 was also identified. Furthermore, transcriptional upregulation of IFNB1 was confirmed by a real time PCR experiment. These data strongly demonstrated HIV-1 cell-to-cell transmission in MT2 cells triggered cGAS-STING pathway and activated IFN gene expression. Meanwhile, the influence of host cell autophagy process to HIV-1 cell-to-cell transmission was also studies. It was found that the induction of autophagy by rapamycin mildly inhibited HIV-1 cell-to-cell transmission; while autophagy inhibitors 3MA, Baf. A and chloroquine also blocked HIV-1 cell-to-cell transmission, except E64D/ pepstatin which inhibits final proteolytic stage of autophagy. Together, our results have provided compelling evidence that HIV-1 cell-to-cell transmission activates IFN and interacts with autophagy system, which might have significant impact on HIV-1 pathogenesis through the host innate immune response and inflammatory signaling.Item Analysis of Antiviral Roles of p53 in the Early Replication of Retrovirus(Albany College of Pharmacy and Health Sciences Theses, 2014) Alghamdi, Faris Saeed '14The tumor suppressor protein p53 has been known as "the guardian of the genome". p53 functions to maintain the host cell genome integrity by arresting cell cycle, activating DNA repair, and inducing apoptosis. Research has demonstrated that retroviruses interact with various host cell proteins involved in the cell cycle regulation and DNA repair machinery. However, the possible influence of p53 in the replication of retroviruses has not been well studied. In order to investigate the role of p53 in retrovirus early replication, Murine leukemia virus (MLV) vector based retroviruses were produced and used to infect human HCT 116 p53+/+ cells and HCT 116 p53-/- cells. MLV was produced by co-transfection of GP2 293 packaging cells by pRetroZsGreen1 and pVSV-G plasmids. Real time PCR quantification showed that the produced retroviruses titer was up to 3.23x108 copies/ml. The infection of MLV was performed in both dividing HCT116 cells and non-dividing HCT116 cells cultured by prolonged serum depletion. The result showed that there were no obvious difference in infection rate, amount of late reverse transcription (RT) product, and circular viral DNA between the dividing HCT116 p53+/+ and dividing HCT116 p53 -/- cells; indicating that p53 does not block the replication of MLV in dividing cells. However, it was found that the infection rate in non-dividing HCT 116 p53-/- (56%) doubled the infection rate in non-dividing HCT116 p53+/+cells (28%). Meanwhile, the amounts of late RT, circular viral 1-LTR, and 2-LTR DNA were significantly higher in non-dividing HCT116 p53-/- compared with non-dividing HCT 116 p53+/+ cells. This result strongly suggests that p53 inhibits MLV early replication in non-dividing cells. It was also evidenced that p53 promotes the formation of viral 1 LTR circular DNA in both dividing and no-dividing cells. Our data implies p53 might be an important host cellular factor in defending retrovirus infection in non-dividing cells.