ROLE OF POLYPEPTIDE pVIII IN BOVINE ADENOVIRUS (BAdV)-3 LIFECYCLE
| dc.contributor.advisor | Tikoo, Suresh K | |
| dc.contributor.committeeMember | Chelico, Linda | |
| dc.contributor.committeeMember | Zhou, Yan | |
| dc.contributor.committeeMember | Zakhartchouk, Alexandre | |
| dc.contributor.committeeMember | Hill, Janet | |
| dc.creator | Gaba, Amit | |
| dc.date.accessioned | 2016-11-01T21:25:39Z | |
| dc.date.available | 2017-11-27T16:32:00Z | |
| dc.date.created | 2016-10 | |
| dc.date.issued | 2016-11-01 | |
| dc.date.submitted | October 2016 | |
| dc.date.updated | 2016-11-01T21:25:39Z | |
| dc.description.abstract | ABSTRACT Bovine adenovirus-3 (BAdV-3) a member of genus Mastadenovirus was first isolated in England from the eye of seemingly healthy cattle (Darbyshire et al., 1965). The linear double stranded genome of BAdV-3 is of 34446 bp in length and is organized into early, intermediate and late regions (Reddy et al., 1998). The mRNAs produced from late region are grouped into seven families (L1-L7) based upon usage of polyadenylation sites (Reddy et al., 1998).The L6 region of BAdV-3 encodes pVIII protein a structural protein associated with hexon connecting the core with the adenovirus capsid (Reddy et al., 1998,Russell., 2009). The objective of the present study was to characterize interaction of pVIII with cellular protein eukaryotic initiation factor 6 (eIF6) and to determine the role of pVIII in the life cycle of BAdV-3. Our initial yeast two-hybrid results indicated that pVIII interacts with cellular protein eukaryotic initiation factor 6 (eIF6). We validated our yeast two-hybrid results using GST pull-down assay, co-immunoprecipitation assay and bimolecular fluorescence complementation (BiFC) assay. Moreover, BAdV-3 late protein pVIII interacts with eIF6 in transfected and BAdV-3 infected cells. Analysis of the interaction of mutant BAdV-3 pVIIIs with eIF6 using co-immunoprecipitation assay identified amino acids 147 to 174 of BAdV-3 pVIII involved in interaction with eIF6. Similar analysis of plasmids expressing mutant eIF6 proteins identified amino acids 44 to 97 of eIF6 involved in interaction with BAdV-3pVIII. The eukaryotic initiation factor 6 is a 245 amino acid protein that is important both for ribosome biogenesis and protein translation (Miluzio et al., 2009). It specifically binds to free 60S ribosomal subunit and prevents its joining with 40S ribosomal subunit (Gartmann et al., 2010,Russell & Spremulli., 1979,Valenzuela et al., 1982). Here, we report a novel interaction between BAdV-3 pVIII and cellular protein eIF6, which appears to alter the formation of functional 80S ribosomes and may modulate the cellular protein translation. The polysome profile analysis of uninfected and BAdV-3 infected MDBK cells indicated that there are more free 60S subunits in BAdV-3 infected cells as compared to uninfected cells suggesting the inhibition in joining of 40S and 60S ribosomal subunits. Comparison of polysome profiles of VIDO GT1 (stable cell line expressing BAdV-3 pVIII) and VIDO DT1 (not expressing BAdV-3 pVIII) cells also revealed the presence of more free 60S and free 40S subunits and reduction in functional 80S ribosomes in pVIII expressing cells compared to VIDO DT1 cells. These results suggest that the presence of pVIII impairs joining of 40S and 60S subunits, which may lead to reduction in formation of functional 80S subunits. To study the biological significance of pVIII-eIF6 interaction, we constructed a recombinant BAdV-3-d147-174, expressing mutant pVIII (containing deletion of domain interacting with eIF6). Analysis of the growth kinetics of BAdV-3-d147-174 suggested that growth of mutant virus was significantly affected. Moreover, CsCl2 gradient purification and TEM analysis revealed the significant decrease in the formation of mature virus particles in BAdV-3-d147-174 infected cells. Analysis of viral gene expression revealed that while the expression of early gene product (DBP) was not affected, the expression of all the analyzed late adenovirus proteins was significantly decreased in the cells infected with BAdV-3-d147-174. However, there was no significant difference in the incorporation of analysed structural proteins between BAdV-304a and BAdV-3-d147-174. These results indicate that abrogating the interaction of pVIII with eIF6 affects expression of adenovirus late proteins, which may result in decreased production of progeny virus. We speculate that interaction of pVIII and eIF6 plays a major role in preferential translation of adenoviral mRNAs during the late phase of infection. Proteolytic maturation involving cleavage of six structural and one non-structural precursor protein is an important aspect of adenovirus life cycle. However, it is not known what role each individual cleavage event plays in determining adenovirus particle stability and infectivity. Analysis of amino acid sequence of BAdV-3 pVIII identified two potential adenovirus protease cleavage sites. Our results suggest that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus cleavage sites. Further, while abrogation of cleavage at only one site does not affect the formation of mature virus, the absence of cleavage at both sites appears lethal for the production of progeny virus. Moreover, the cleavage of pVIII at both potential cleavage sites is a major contributing factor in producing stable BAdV-3 particles. Together, these studies provide evidence that proteolytic maturation of pVIII is essential for correct virus assembly and stability of the adenovirus particle. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10388/7553 | |
| dc.subject | Bovine adenovirus-3 | |
| dc.subject | eIF6 | |
| dc.subject | protease | |
| dc.subject | pVIII | |
| dc.subject | cleavage | |
| dc.title | ROLE OF POLYPEPTIDE pVIII IN BOVINE ADENOVIRUS (BAdV)-3 LIFECYCLE | |
| dc.type | Thesis | |
| dc.type.material | text | |
| local.embargo.terms | 2017-11-01 | |
| thesis.degree.department | Veterinary Microbiology | |
| thesis.degree.discipline | Veterinary Microbiology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) |