Amino Acid Exchanges in the Putative Nuclear Export Signal of Adenovirus Type 5 L4-100K Severely Reduce Viral Progeny due to Effects on Hexon Biogenesis
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Fig 1Negative selection against H5pm4165 by homologous recombination in H1299 cells. (A) Multiple amino acid sequence alignment of L4-100K polypeptides from different human Ad types classified into 6 subgroups (A to F) compared to the NES consensus sequence (Φ: hydrophobic amino acid residue; X: any amino acid residue). Numbers in brackets refer to the number of amino acid residues in the respective L4-100K protein. Hydrophobic residues that are identical in all types are shown in light gray boxes. The conserved hydrophobic residue is marked in a black box. (B) Nucleotide sequence of wt H5pg4100 and mutant H5pm4165 viruses. Numbers refer to positions of nucleotides in the wt L4-100K DNA sequence. Hydrophobic amino acids encoded by the underlined codons are shown below. (C) Sequencing data of an H5pm4165 virus stock generated by cotransfecting Ad2-100K plasmid. Arrowheads indicate the mixed sequence (N) of nucleotides at the positions of inserted mutations. Asterisks denote the mixed sequence of nucleotide residues that differ between Ad2 and Ad5 L4-100K DNA sequences. (D) The purity of the H5pm4165 virus stock was tested by PCR amplification and subsequent digestion of the purified amplicon with the restriction enzyme SacI. The DNA restriction pattern was analyzed on an agarose gel and compared to those of similarly treated wt (pH5pg4100) and mutant L4-100K (pH5pm4165) bacmid DNA.
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Fig 2Effects of amino acid substitutions in L4-100K on protein synthesis and virus growth. (A) Viral protein synthesis. A549 cells were infected with the wt or mutant virus at a multiplicity of 10 focus-forming units (FFU) per cell. Total cell extracts were prepared at indicated times after infection. Proteins (25 μg samples) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with mouse MAb B6-8 (E2A-72K), mouse MAb 2A6 (E1B-55K), rat MAb 6B10 (L4-100K), and anti-Ad5 rabbit polyclonal antiserum L133 and with anti-PRMT1 rabbit polyclonal antibody as a loading control. Bands corresponding to viral late proteins hexon (II), penton (III), and fiber (IV), as well as capsid-associated proteins (VI, VII, VIII, and IX), are indicated on the right. (B) Virus yield. A549 cells were infected with wt H5pg4100 or H5pm4165 at indicated multiplicities. Viral particles were harvested at indicated time points (h.p.i.) and virus yield was determined by quantitative E2A-72K immunofluorescence staining on K16 cells. The results represent the averages from three independent experiments. Error bars indicate the standard errors of the means.
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Fig 3Effect of L4-100K NES mutation on hexon localization, binding, and trimerization. (A, B) Intracellular localization of L4-100K and hexon (A) or pVI (B) in virus-infected cells. A549 cells were infected with H5pg4100 and H5pm4165 viruses at a multiplicity of 10 FFU per cell and fixed at 36 h.p.i. Cells were labeled with anti-L4-100K rat MAb 6B10 (α-L4-100K) and anti-hexon rabbit polyclonal serum (A; α-hexon) or anti-protein VI rabbit MAb pVI (B; α-pVI), plus Cy5- and Cy3-conjugated secondary antibodies, respectively. Representative anti-L4-100K (green; a and d) and antihexon or anti-pVI (red; b and e) antibody staining patterns are shown. Overlays of DAPI (4′,6′-diamidino-2-phenylindole) staining (blue) with the green and red images are shown in panels c and f (merge). Nuclei visualized using DAPI are indicated by a dashed line in all panels. Arrowheads in the images indicate L4-100K, hexon, and pVI cytoplasmic aggregates and/or L4-100K/hexon colocalization. (C) Interaction between hexon and L4-100K. H1299 cells were transfected with wt (pwt) or NES pTL-flag-100K (pNES) plasmids and infected with H5pg4100 virus (10 FFU/cell) 6 h after transfection. Total cell extracts were prepared from noninfected (mock) and transfected-infected cells 30 h.p.i. Aliquots of 25 μg of lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-flag MAb M2, antihexon rabbit polyclonal serum, and anti-PRMT1 rabbit polyclonal antibody as a loading control. The same lysates were used for immunoprecipitation (IP) with anti-Flag MAb. The immunocomplexes were separated by SDS-PAGE and analyzed by immunoblotting using the antihexon rabbit polyclonal serum. (D) Hexon trimerization in virus-infected cells. A549 cells were infected with H5pg4100 and H5pm4165 viruses at a multiplicity of 1 FFU per cell and harvested at 48 h.p.i., and total cell extracts were prepared under native conditions. Proteins (25-μg samples for hexon and 10-μg samples for L4-100K and β-actin) were separated by SDS-PAGE and subjected to immunoblotting using antihexon rabbit polyclonal antiserum, anti-L4-100K rat MAb 6B10, and anti-β-actin mouse MAb AC-15. Bands corresponding to hexon monomers and/or trimers are indicated on the right.
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Fig 4Mapping hexon binding regions in L4-100K. (A) Schematic diagram showing the location of the putative nuclear export signal (NES) in the L4-100K protein. GST-tagged fusion fragments of L4-100K (F1-F4) are depicted below the protein map. Numbers refer to amino acid residues of Ad5 L4-100K. (B) GST and GST-100K fragments were purified from E. coli using glutathione-Sepharose. Purified proteins were subjected to SDS-PAGE and Coomassie brilliant blue staining. M, molecular-weight marker. (C) H5pg4100-infected A549 cell lysates were incubated with GST or GST fusion fragments. Aliquots of 25 μg from the cell lysates were used as input. GST-protein complexes were isolated with glutathione-Sepharose and subjected to SDS-PAGE and Western blotting with antihexon antibody.
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