ROLE OF MITOCHONDRIA DURING BOVINE ADENOVIRUS 3 INFECTION
Bovine adenovirus (BAdV) -3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Mitochondria are multifunctional organelles, which are involved in various functions of the cell including but not limited to energy production, aging, regulation of cell cycle, anti viral responses. Thus, this makes them strategic targets for many pathogens. Although a number of viruses affect the structure and function of mitochondria, the effect of BAdV-3 infection on these organelles has not been well characterized. The aim of the present study was to ascertain the pathological effects of BAdV-3 infection on host mitochondria and the role of BAdV-3 encoded proteins in modulating mitochondrial functions. Electron microscopy analysis revealed extensive damage to the inner mitochondrial membrane characterized by dissolution of cristae and amorphous appearance of mitochondrial matrix with little or no damage to the outer mitochondrial membrane. There were fewer cristae with altered morphology. Patches of protein synthesis machinary around mitochondria were observed at 12 hrs post infection. At 24 hrs post-infection, extensive damage to mitochondria was evident throughout the infected cell. ATP production, mitochondrial Ca2+ and mitochondrial membrane potential (MMP) peaked at 18 hrs post-infection but decreased significantly at 24 hrs post-infection. This decrease coincided with increased production of superoxide (SO) and reactive oxygen species (ROS), at 24 hrs post-infection indicating acute oxidative stress in the cells and suggesting a complete failure of the cellular homeostatic machinary. Sequence analysis of BAdV-3 proteins revealed the presence of potential mitochodria localization signals (MLS) in 52K, VII, 33/22K and IVa2. Western blot analysis of isolated mitochondrial fractions suggested that all these proteins are localized in the mitochondria. However, a more stringent proteinase K assay confirmed the presence of 52K and pVII in the mitochondria suggesting that the other observed proteins were loosely attached to the surface of the mitochondria or may simple co-purify with the mitochondrial fraction. The presence of potential MLS in 52K and pVII was confirmed by localization of EYFP (Enhanced Yellow Fluorescent Protein; a predominantly cytoplasmic protein), when fused to MLS of pVII or 52K, to mitochondria of transfected cells. Expression of pVII in transfected cells showed an increase in MMP and ATP production, and increased sequestration / retention of mitochondrial Ca2+ in the cells. However, there was no increase in reactive oxygen species (ROS) / superoxide (SO) production in pVII transfected cells indicating that pVII acts as an antiapototic protein. In contrast, expression of 52K in transfected cells significantly increased ROS/SO production with no significant change in ATP production, mitochondrial Ca2+ or MMP indicating that 52K alone causes an oxidative stress in cells following infection and causes apoptosis. In conclusion, these results reveal an intricate relationship between Ca2+ homeostasis, the ATP generation ability of cells, SO and ROS production and regulation of MMP following infection by BAdV-3 or transfection of the cells with plasmid DNAs expressing pVII & 52K. While pVII appears to contribute to the survival of the cells during virus replication, 52K is involved in the death of the infected cells and thus may help in release of progeny virus.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorTikoo, Suresh K.
CommitteeHill, Janet; Singh, Jaswant; Misra, Vikram; Zakhartchouk, Alexander
Copyright DateNovember 2011
Bovine Adenovirus 3
Reactive Oxygen Species
Mitochondrial Membrane Potential
Reactive Nitrogen Species