The Role of Heat Shock Proteins (HSP) in Rift Valley Fever Virus Infection
| dc.contributor.advisor | Hakami, Ramin M. | |
| dc.contributor.author | Benedict, Ashwini | |
| dc.creator | Benedict, Ashwini | |
| dc.date | 2013-07-12 | |
| dc.date.accessioned | 2013-12-03T20:02:02Z | |
| dc.date.available | 2013-12-03T20:02:02Z | |
| dc.date.issued | 2013-12-03 | |
| dc.description.abstract | Rift Valley Fever Virus (RVFV) in the family Bunyaviridae is an emerging infectious pathogen and the causative agent of Rift Valley Fever, a zoonotic arthropod-borne disease characterized by potentially fatal hemorrhagic fever in humans and high abortion rate in pregnant ruminants. RVFV is a negative-sense tripartite RNA virus that encodes a complement of several proteins, including the structural protein N, the RNA-dependent RNA polymerase L, and an essential non-structural virulence factor NSs. RVFV is a category A priority pathogen for which there are currently no approved vaccines or therapeutics. Therefore, it is imperative to understand the range of critical host-pathogen interactions that occur. In this research, the importance of host HSPs in RVFV infection is demonstrated. Proteomic profiling showed that host HSPs are among the over-represented protein families within the RVFV virions. Time-of-addition studies with HSP90 inhibitor 17-AAG, HSP70 inhibitor KNK437 and general HSP inhibitor BAPTA-AM demonstrated that the HSP effects occur early, manifesting within hours after infection. Consistent with these results, real-time studies by monitoring luciferase signal from cells treated with HSP inhibitors and subsequently infected with luciferase-expressing virus, showed that 17-AAG causes a significant decrease in viral load in early infection (4 and 8 hours p.i.). These findings suggest HSP effects on viral replication and/or transcription. Specific effects of 17-AAG on RVFV RNA levels were also demonstrated using qRT-PCR analysis. Treatment with 17-AAG caused a reduction of viral RNA levels at the following p.i. times: 4hr, 6hr, 8hr, 10hr, and 24hr. Specific effects of HSP inhibitors on RVFV protein levels were also demonstrated using Western blot analysis. Interestingly, while 17-AAG significantly decreased both viral NSs and N-protein levels, KNK437 caused a significant decrease only in NSs, indicating that the HSPs act through distinct functional mechanisms. For 17-AAG, the time course of its effect on the levels of N, L, and NSs were measured by Western blot analysis matching the time-points analyzed with qRT-PCR. A reduction in the protein levels were observed at the earliest time of detection (4hr p.i. for L and NSs, and 8hr p.i. for N). Confocal imaging of HSP90 with N-Protein did not show colocalization, suggesting that this pair of viral-host proteins do not form a complex with each other. Treatment with the proteasome inhibitor MG-132 showed that at least part of HSP90 mechanism is by stabilizing RVFV proteins and preventing their rapid degradation. The HSP effects on viral infection were observed in different cells types (Vero cells and HepG2 cells) and also with both the attenuated strain of RVFV (MP-12) and the fully virulent strain (ZH-501). These studies provide much-needed insight into RVFV-host interactions. As 17-AAG and several other HSP90 inhibitors are already in clinical trials for cancer treatment, there is the exciting potential of repurposing them to treat RVF. Given the demonstrated role of HSP90 for other RNA viruses, the strategy of targeting HSP90 also presents broad spectrum therapeutic options for other RNA viruses. | |
| dc.identifier.uri | https://hdl.handle.net/1920/8623 | |
| dc.language.iso | en | |
| dc.subject | Heat shock proteins | |
| dc.subject | RVFV | |
| dc.subject | Rift Valley Fever Virus | |
| dc.subject | HSP90 | |
| dc.subject | HSP | |
| dc.subject | 17-AAG | |
| dc.title | The Role of Heat Shock Proteins (HSP) in Rift Valley Fever Virus Infection | |
| dc.type | Thesis | |
| thesis.degree.discipline | Biology | |
| thesis.degree.grantor | George Mason University | |
| thesis.degree.level | Master's | |
| thesis.degree.name | Master of Science in Biology |