Bovine adenovirus type 3 (BAdV-3) is a non-enveloped icosahedral particle which contains a double stranded DNA genome. The genome of BAdV-3 is organized into early, intermediate and late regions. The late region is organized into seven regions L1-L7 (Reddy et.al., 1998). The L6 region of late transcription unit of BAdV-3 encodes one of the non structural protein named 33K protein. The objective of the present study was to characterize the 33K protein and to identify the viral/cellular proteins involved in the interaction with 33K protein.<p> The RT-PCR analysis revealed the presence of spliced and unsliced mRNAs encoding 33K and 22K proteins respectively in BAdV-3 infected cells. The 33K and 22K proteins share a N-terminus region of 138 amino acids. To determine the specificity of these two proteins, rabbit polyclonal antiserum was raised against peptides representing unique C- terminal regions of the proteins. Anti-33Kp serum detected two major proteins of 42 kDa and 22 kDa and five minor proteins of 39kDa, 35kDa, 29kDa, 25kDa and 19kDa in BAdV-3 infected cells or 33K transfected cells. Similarly, anti-22Kp serum detected three proteins of 41kDa, 39kDa and 37kDa in BAdV-3 infected cells. However, a protein of 39kDa and 37kDa was detected in 22K (having splice sites removed) transfected cells. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells and is involved in stimulating the transcription from major late promoter. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 and amino acid 204-231 are required for nuclear localization and MLP transactivation.<p> The adenovirus 33K protein appears to be a multifunctional protein performing different role in viral infection. Earlier study has shown that the 33K protein plays a role in viral capsid assembly and efficient capsid DNA interaction in BAdV-3 (Kulshreshtha et.al., 2004). The involvement of 33K protein in different steps of adenovirus replication may require protein protein interaction. Using 33K protein as bait in yeast two hybrid system, open reading frames (ORFs) of BAdV-3 were screened for the potential interactions with 33K protein. The 33K protein showed specific interactions with two late viral proteins- 100K and protein V (pV). The yeast two hybrid findings were validated by in vitro binding using <i>in vitro</i> synthesized transcription-translation products. It was demonstrated that the interaction of 33K with 100K and pV takes place during BAdV-3 infection. The stretch of amino acids 81-120 and 161-200 in 33K protein were involved in the interaction with pV and 100K protein.<p> For screening the cellular interactions, the 33K protein was used as a bait to screen bovine retina cDNA library. The yeast two hybrid screening revealed that the 33K protein appears to interact with bovine presenilin-1-associated protein / mitochondrial carrier homolog 1 (BoPSAP / BoMtch1) and bovine microtubule associated protein (BoMAP). However, subsequent analysis by various <i>in vitro</i> and <i>in vivo</i> assays could only confirm the interaction between 33K protein and BoPSAP/BoMtch1. In addition, the 33K protein was also shown to be colocalized with BoPSAP in mitochondria. Based on these observations, it may be possible that 33K protein may play an anti-apoptotic by interacting with BoPSAP since the human homolog of PSAP has been known to induce apoptosis.

Bovine adenovirus type 3 (BAdV-3) is a non-enveloped icosahedral particle which contains a double stranded DNA genome. The genome of BAdV-3 is organized into early, intermediate and late regions. The late region is organized into seven regions L1-L7 (Reddy et.al., 1998). The L6 region of late transcription unit of BAdV-3 encodes one of the non structural protein named 33K protein. The objective of the present study was to characterize the 33K protein and to identify the viral/cellular proteins involved in the interaction with 33K protein.<p> The RT-PCR analysis revealed the presence of spliced and unsliced mRNAs encoding 33K and 22K proteins respectively in BAdV-3 infected cells. The 33K and 22K proteins share a N-terminus region of 138 amino acids. To determine the specificity of these two proteins, rabbit polyclonal antiserum was raised against peptides representing unique C- terminal regions of the proteins. Anti-33Kp serum detected two major proteins of 42 kDa and 22 kDa and five minor proteins of 39kDa, 35kDa, 29kDa, 25kDa and 19kDa in BAdV-3 infected cells or 33K transfected cells. Similarly, anti-22Kp serum detected three proteins of 41kDa, 39kDa and 37kDa in BAdV-3 infected cells. However, a protein of 39kDa and 37kDa was detected in 22K (having splice sites removed) transfected cells. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells and is involved in stimulating the transcription from major late promoter. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 and amino acid 204-231 are required for nuclear localization and MLP transactivation.<p> The adenovirus 33K protein appears to be a multifunctional protein performing different role in viral infection. Earlier study has shown that the 33K protein plays a role in viral capsid assembly and efficient capsid DNA interaction in BAdV-3 (Kulshreshtha et.al., 2004). The involvement of 33K protein in different steps of adenovirus replication may require protein protein interaction. Using 33K protein as bait in yeast two hybrid system, open reading frames (ORFs) of BAdV-3 were screened for the potential interactions with 33K protein. The 33K protein showed specific interactions with two late viral proteins- 100K and protein V (pV). The yeast two hybrid findings were validated by in vitro binding using <i>in vitro</i> synthesized transcription-translation products. It was demonstrated that the interaction of 33K with 100K and pV takes place during BAdV-3 infection. The stretch of amino acids 81-120 and 161-200 in 33K protein were involved in the interaction with pV and 100K protein.<p> For screening the cellular interactions, the 33K protein was used as a bait to screen bovine retina cDNA library. The yeast two hybrid screening revealed that the 33K protein appears to interact with bovine presenilin-1-associated protein / mitochondrial carrier homolog 1 (BoPSAP / BoMtch1) and bovine microtubule associated protein (BoMAP). However, subsequent analysis by various <i>in vitro</i> and <i>in vivo</i> assays could only confirm the interaction between 33K protein and BoPSAP/BoMtch1. In addition, the 33K protein was also shown to be colocalized with BoPSAP in mitochondria. Based on these observations, it may be possible that 33K protein may play an anti-apoptotic by interacting with BoPSAP since the human homolog of PSAP has been known to induce apoptosis.

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. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

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. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

Bovine adenovirus (BAdV)-3, a Mastadenovirus was isolated from the healthy and sick cattle (Darbyshire et al., 1965; Zhu et al., 2011). Like other adenoviruses, BAdV-3 replication is characterized by the temporally regulated expression of genes characterized by early, intermediate and late gene expression. Genus-common, non-structural protein 100K is encoded by late region L6 of BAdV-3. The objective of the present study was to characterize the BAdV-3 100K protein and identify cellular and viral proteins interacting with 100K. Although BAdV-3 100K encoded as 850 amino acid polypeptide (Reddy et al., 1998), rabbit antisera raised against peptides representing N-terminus or C-terminus recognized a protein of 130 kDa at 12-24 hrs post infection, and proteins of 130 kDa, 100 kDa, 95 kDa and 15 kDa at 36-48 hrs post infection. The 100K appeared to be localized to the nucleus and cytoplasm of BAdV-3 infected cells. In contrast, 100K localized predominantly to cytoplasm of transfected cells. However, BAdV-3 infection of cells transfected with 100K-EYFP expressing plasmid detected fluorescent protein in nucleus of the cells suggesting that another viral protein may be required for the nuclear localization of 100K. Using yeast two-hybrid and GST pull-down assays, 100K protein was shown to interact with BAdV-3 33K protein. These results were validated using bimolecular fluorescence complementation (BiFC) assay. Although, 100K protein interacts with 33K protein, co-expression of both proteins in transfected cells did not alter the cytoplasmic localization of 100K. Using GST-pull down assay and BiFC assay, 33K interacting region of 100K was localized to a stretch of 13 amino acids (624-637). Repeated attempts were not successful in rescuing a recombinant BAdV-3 expressing mutant 100K (containing deletion of amino acids 624-637). The interaction of cellular protein(s) with 100K was determined by mass spectrometric analysis of immunoprecipitated 100K. Mass spectrometry of immunoprecipitate obtained by immunoprecipitating 100K protein from BAdV-3 infected cells harvested at 48 hrs post infection identified six proteins including dynein light chain (DYNLT)1. The initial identified interaction of 100K with DYNLT1 was confirmed by the yeast two-hybrid assay, co-immunoprecipitation assay and BiFC assays. Furthermore, DYNLT1 interacting domain of 100K protein of BAdV-3 was found to be located between 499-587 amino acids. Co-expression of BAdV-3 100K-EY fusion protein with myc epitope tagged DYNLT1 protein did not alter the localization of 100K-EY fusion protein. The investigation into the differences in the subcellular localization of the 100K protein in the transfected and infected cells lead to identification of the cleavage by adenoviral protease. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (amino acid 740-745 and 781-786) in transfected or BAdV-3 infected cells. Although protease encoded by human adenovirus (HAdV)-5 or porcine adenovirus (PAdV)-3 also cleaved BAdV-3 100K at potential identified protease cleavage sites, no such cleavage of 100K encoded by HAdV-5 or PAdV-3 could be detected in cells expressing virus specific protease. Successful isolation of recombinant BAdV-3 expressing mutant protease (substitution of alanine for glycine in potential protease cleavage site) suggested that cleavage of BAdV-3 100K by viral protease is not essential for viral replication. However, further analysis observed less virus in the supernatant of cells infected with mutant BAdV-3 compared to WT BAdV-3 suggesting a possible role for cleaved C-terminal fagment in lysis of infected cells. Co-expression of BAdV-100K with other late viral proteins suggested that the 100K-EYFP fusion protein localized to the nucleus in cells co-expressing BAdV-3 protease-DsRed fusion protein. Interestingly, only C-terminal cleaved fragment of 100K localizes to the nucleus in BAdV-3 protease expressing cells. Further analysis suggested that C-terminal fragment localizing to the nucleus contains a bipartite nuclear localization signal, which is recognized by importin α3. Our results suggest that the N-terminal part of 100K may be retained in the cytoplasm by interaction with Tctex1 (DYNLT1). Our study provides for the first time a plasmid co-transfection system for the study of the protease cleavage of viral proteins. Moreover, this is the first report of cleavage of any non-structural viral proteins by adenoviral protease in infected cells.