Leucine residues in conserved region of 33K protein of bovine adenovirus – 3 are important for binding to major late promoter and activation of late gene expression

AbstractThe L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37kDa in BAdV-3 infected cells and one protein of 42kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP

Conserved Arginines of Bovine Adenovirus-3 33K Protein Are Important for Transportin-3 Mediated Transport and Virus Replication

<div><p>The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion.</p></div

Sub cellular localization of 33K proteins.

<p>(<b>A</b>) <i>Schematic representation of BAdV-3 33K fusion proteins</i>. The number above the box denote the amino acid number for 33K protein. Thick box represent BAdV-3 DNA; hollow box represents EYFP DNA; stripped box represents GFP DNA; dotted box represents β-gal DNA. Thin lines represent deleted regions. The name of the individual mutant protein is indicated on the left of the panel. The name of the individual plasmid is indicated on the right of the panel. (<b>B</b>). <i>Immunofluorescence</i>. Monolayers of MDBK cells (panel A) infected with BAdV-3. At 24 hrs post infection, the cells were analysed by indirect immunofluorescence by staining with anti-33Kp serum, Cy 2 conjugated goat anti-rabbit serum and DAPI. Similarly, monolayers of HeLa cells (panels B to P) were transfected with individual plasmid DNA expressing indicated 33K fusion proteins and analysed at 48 hrs post transfection. Finally, the cells were stained with DAPI and analyzed by direct fluorescence. The name of the protein is indicated on the right of the panel.</p

Analysis of BAdV-3 33K.

<p>(<b>A</b>). <i>Schematic representation of BAdV-3 33K</i>. The coding sequences shared by 33K and 22K (box with Pattern) or specific for 33K (hollow box) are depicted. The spliced region in 33K is represented by dots flanked by splice donor\acceptor sites. The wild-type (GT…AG) and mutated (GC…CG) splice acceptor/donor sites are depicted. The nucleotide numbers of BAdV-3 genome shown are according to Gene bank accession # AF030154 residues are underlined. The star represents the stop codon. The name of the encoded protein is depicted on the right of the panel. The name of the plasmid is depicted on the left of the panel. (<b>B</b>). <i>Western blot analysis</i>. Protein lysates of BAdV-3 infected MDBK cells or plasmid DNA transfected 293T cells were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-33Kp serum. The position of the molecular weight markers (M) in kDa is shown to the left of the panel. Arrows on the right of the panel indicate the position of the identified protein in kDa.</p

Sub cellular localization of mutant 33Ksr protein.

<p>(<b>A</b>) <i>Amino acid homology of BAdV-3 33K like proteins</i>. Alignment of deduced amino acid sequences of BAdV-3 33K homologs with those of canine adenovirus (CAdV)-2 (GeneBank Accession # AC_000020), HAdV-2 (GeneBank Accession # AC_000007), HAdV-5 (GeneBank Accession # AC_000008), HAdV-35 (GeneBank Accession # AC_000019). The potential RS domain is overlined. RS repeat residues are indicated (*). (<b>B</b>) <i>Schematic representation of BAdV-3 33K fusion proteins</i>. The number above the thick box denote the amino acid number for 33K protein. Thick box represent BAdV-3 DNA. Dotted lines represent deleted regions. The name of the individual mutant protein is indicated to the left of the panel. The name of the individual plasmid is indicated on the right of the panel. (<b>C</b>) <i>Western blot</i>. Proteins from lysates of 293T cells transfected with indicated plasmids were separated by 15% SDS-PAGE, transferred to nitrocellulose and probed in Western Blot using anti-33K serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101216#pone.0101216-Kulshreshtha2" target="_blank">[15]</a>. (<b>D</b>) <i>Immunofluorescence</i>. Monolayers of HeLa cells were transfected with indicated individual plasmid DNA and analysed at 48 hrs post transfection by indirect immunofluorescence by staining with anti-33Kp serum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101216#pone.0101216-Kulshreshtha2" target="_blank">[15]</a>, Cy 3 conjugated goat anti-rabbit serum and DAPI and analyzed by direct fluorescence. The name of the plasmid is indicated on the left of the panel.</p

<i>In-vitro</i> nuclear import assays.

<p>MDBK cells were permeabilized with digitonin and incubated with (<b>A</b>) GST-NLS-GFP (panels a,b,c,d,e,f,g,h,I,j) or (<b>B</b>) GST-33K (panels a,b,c,d,e,f,g,h,I,j). Import reactions were carried out in the presence (panel a) or absence of rabbit reticulocyte lysates (panel b), absence of ATP generating system (panel c), in the presence of the dominant negative mutant RanQ69L (panel d), in the presence of wheat germ agglutinin (WGA) (panel e), incubation at 40C (panels f), in the presence of inhibitory peptides IBB Impα (panel h), IBBrpL23α (panel g) Ycbp80 (panels i), SR1 (panel j).</p

<i>In-vitro</i> interaction of 33K with transport receptors.

<p>(<b>A</b>) Purified GST fusions of importin -α1 (lane 7), -α3 (lane 6), -α5 (lane 5), -α7 (lane 4) or importin -β1 (lane 2) along with GST alone were incubated with <i>in -vitro</i> transcribed and translated, [³⁵S]-labeled 33K. Input [³⁵S]-labeled 33K (lane 1). (<b>B</b>) Purified GST fusions of importin-α5 (lanes 2,5,8) or TRN-SR2 (lanes 1,4,7) along with GST alone (lane 10) were incubated with <i>in-vitro</i> transcribed and translated [³⁵S]-labeled 33K (lanes 8,10), 33Kd6 (lanes 4,5) or 33Kd6b proteins (lanes 1,2). Input [³⁵S]-labeled 33K (lane 9), [³⁵S]-labeled 33Kd6 (lane 6), [³⁵S]-labeled 33Kd6b (lane 3) (<b>C</b>) Purified GST-TRN-SR2 fusion protein (lanes 2,3) or GST alone (lane 2) were incubated with <i>in-vitro</i> transcribed and translated [³⁵S]-labeled 33K (lane 2,3). Input [³⁵S]-labeled 33K (lane 1). (<b>D</b>) Purified GST- importin-α5 fusion protein (lane 1), GST-TRN-SR2 fusion protein (lane 2) or GST alone (lane 4) were incubated with <i>in-vitro</i> transcribed and translated [³⁵S]-labeled 33Ksr protein (lanes1, 3). Input [³⁵S]-labeled 33K (lane 3). Samples from (A), (B), (C) and (D) were pulled down with glutathione Sepharose beads, separated by 10% SDS-PAGE and visualized using a phosphor screen. 10% of the input [³⁵S]-protein was run as a control.</p

L6 33K and 22K.

<p>(<b>A</b>) <i>Sequence of 33K and 22K</i>. The nucleotide numbers of BAdV-3 genome shown are according to Gene bank accession # AF030154. The changed nucleotide residues in 33K and 22K are shown in bold. The substituted amino acid in 33Ksr or 22Kw is shown in bold and underlines. (<b>B</b>). <i>Schematic representation of plasmid pFBAV33Ksr</i>. BAdV-3 genome (dotted pattern box). The thin line represented deleted E3 region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101216#pone.0101216-Zakhartchouk1" target="_blank">[45]</a>. The substituted amino acids (glycines) of 33K in BAdV-3 genome are underlined. The numbers represent the amino acid numbers of 33K. CMV (human cytomegalovirus immediate early promoter); EYFP (enhanced yellow fluorescent protein). Arrows represent the direction of transcription. The coding sequences shared by 33K and 22K (box with pattern) or specific for 33K (hollow box) and 22K (black box) are depicted. The spliced region in 33K is represented by dots flanked by splice donor\acceptor sites. The wild-type (GT…AG) and mutated (GC…CG) splice acceptor/donor sites are depicted. The numbers of the top denote amino acid numbers. (<b>C</b>) <i>Complementation of pFBAV33Ksr genome</i>. The VIDO DT1 cells were co transfected with indicated plasmids and the fluorescent focus forming units were counted at indicated days post transfection. The numbers on the X- axis denote the days post transfection.</p

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 μg of CpG-ODN or saline (n=~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n=3‐4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 μg and 50 μg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1×104 or 1×105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 μg and 50 μg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity