THE ROLE OF BOVINE ADENOVIRUS-3 PROTEIN V (pV) IN VIRUS REPLICATION
Bovine adenovirus type 3 (BAdV-3), which is a non-enveloped icosahedral particle with a double-stranded DNA genome of 34,446 base pair, has been developed as a vaccine vector. It belongs to mastadenovirus genus in adenoviridae family. Like other mastadenovirus members, BAdV-3 genome is composed of early, intermediate and late regions. The seven late regions (L1-7) of BAdV-3 genome all belong to one major late transcription unit (MLTU), which is controlled by the major late promoter (MLP). L2 region encodes only one protein V (pV), which is a core protein and functions as a bridge between the viral capsid and DNA genome. It also has been shown to interact with cellular protein p32, which shuttles between nucleus and mitochondria, and redistribute nucleolar proteins B23 and nucleolin. Both of the cellular protein interaction and redistribution suggest that pV may have other functions in viral replication rather than just being a structural protein. Therefore, the objective of this study is to characterize pV, elucidate the functions of pV in viral replication and identify the viral/cellular proteins interacting with pV. The open reading frame of BAdV-3 pV was confirmed by DNA sequencing analysis to be shown to correspond to 423 amino acids rather than 412 amino acids reported previously. A 55 kDa protein indicating pV was detected by Western blot using anti-pV sera at 24 to 48 hours post-infection in BAdV-3 infected cells, and the same sized protein was detected in pV expressing plasmid transfected cells. pV was distributed in the nucleus, especially in the nucleolus in both infected and transfected cells. Analysis of BAdV-3 pV protein sequence by protein analysis program “PredictProtein” predicted potential motifs act as nuclear localization signals. However, indirect immunofluorescence result suggested that amino acids 21-50 and 380-389 are essential for the nucleolar localization of pV. Further analysis identified three motifs, which are essential for amino acids 21-50 mediated nucleolar localization of pV. GST pulldown assay identified pV nuclear localization was mediated by the importin α/β pathway since pV was detected to interact with importin α3 rather than other nuclear transport factors. Three nuclear localization signals (NLS) were detected in pV by sequential deletion and confocal microscopy. Deletion of all of these NLSs abrogates not only pV nuclear localization but also its interaction with import α3. To determine the function of pV nucleolar localization signals (NoLSs) in BAdV-3 replication, we isolated recombinant BAdV-3s, BAV.pVd1 (deletion of amino acids 21-50), BAV.pVm123 (containing substitutions of basic residues of all three motifs of amino acids 21-50), BAV.pVd3 (containing deletion of amino acids 380-389) and BAV.pVd1 d3 (deletion of amino acids 21-50 and 380-389). Analysis of mutant BAdV-3s suggested that the deletion of single pV NoLS did not influence the replication of BAdV-3 in vitro. However, deletion of both NoLSs rendered BAV.pVd1d3 replication incompetent in non-pV expressing cells. Moreover, the double, but not single, deletion of pV NoLSs, decreased late viral protein expression, viral particle assembly in MDBK cells and viral thermostablity. These results demonstrate that pV contains two NoLSs, which are essential for BAdV-3 replication. To elucidate the biological significance of pV in BAdV-3 replication, a plasmid pUC304A.dV, which contains BAV304a genome except pV gene was replaced by an SbfI site, was constructed and used to transfect VIDO DT1 (cotton rat lung [CRL] cells expressing I-SceI) cells. However, pV deleted BAdV-3 cannot be rescued in this non-pV expressing cells, suggesting pV is indispensible for BAdV-3 replication. Therefore, a pV expressing CRL cell line (CRL.pV) was constructed to support the pV deleted recombinant BAdV-3 (BAV.dV) replication in vitro. Protein analysis revealed that deletion of pV from BAdV-3 genome decreased viral late proteins expression such as pX, 100K and pVII. Electron microscopy analysis revealed that pV deletion results in the dramatic reduction in viral assembly. Moreover, thermostablity assay suggested that the viral thermostablity was significantly decreased without pV incorporation. To determine viral/cellular proteins interacting with pV, an eight amino acids Strep-tag was introduced between pV amino acids 18EI19 to construct a recombinant BAdV-3 with strep-tagged pV. Although expression of viral late proteins was decreased to some extent, the introduction of Strep-tag in pV did not influence viral growth characteristics significantly. Mass spectrometry analysis identified many viral/cellular proteins can interact with pV. The interaction of pV with nucleolin was confirmed by Co-immunoprecipitation (Co-IP) and co-localization. Further analysis indicated that deletion of pV NoLSs did not abrogate pV interaction with nucleolin, suggesting that pV NoLSs are not binding sites for nucleolin. Future work remains to elucidate the function of nucleolin in BAdV-3 replication.
Adenovirus, pV, nuclear localization signal, nucleolar localization signal, nucleolin
Doctor of Philosophy (Ph.D.)
Western College of Veterinary Medicine