Fig 1.
Cre-mediated recombination of the pVII gene from the Ad5-VII-loxP viral genome.
A) Schematic of the Ad5 L2 coding region. Open reading frames that encode the proteins penton (III), pVII, V, and X/μ are depicted in colors. LoxP sites are indicated by upward red arrows. The downward arrow indicates infection of a Cre recombinase-expressing cell line with Ad5-VII-loxP. Cre-mediated recombination leads to excision of the pVII gene with the maintenance of a single lox site. (B) Southern blot analysis of total cell DNA isolated from infections of either 293 cells (–, lanes 2 and 3) or 293 cells expressing Cre recombinase (+, lanes 4 and 5) with the Ad5-VII-loxP virus. Cells were harvested at 48 (lanes 2 and 4) or 72 (lanes 3 and 5) hr post-infection (hpi) and DNAs were analyzed by Southern blot. U represents intact (unfloxed) pVII gene and F represents the recombined (floxed) pVII gene. DNA size markers are indicated on the left (lane 1). (C) Western blot analyses of lysates from 293 cells (–) and 293 cells expressing Cre (+) infected with the Ad5-VII-loxP virus. Ad5 L2 proteins were detected with specific antibodies as described in Supplemental Information. Cellular alpha-tubulin (α-tubulin) was used as a loading control.
Fig 2.
The absence of pVII does not preclude virus particle assembly and genome packaging.
(A) CsCl equilibrium gradient centrifugation of virus particles isolated from isolated from 293 cells (Cre–) or 293 cells expressing Cre recombinase (Cre+) infected with the Ad5-VII-loxP virus. Virus particles from both infections banded on the gradient at 1.34 g/cc, the density of mature Ad5 virions. (B) Electron microscopy images of negative stained CsCl-purified virions isolated from 293 cells (Cre–) or 293 cells expressing Cre recombinase (Cre+) infected with wild-type Ad5 (WT) or the Ad5-VII-loxP virus. Bar inset represents 50nm. (C) Coomassie blue stain analysis of the protein composition of CsCl-purified virions isolated from 293 cells infected with wild-type Ad5 (WT) or the Ad5-VII-loxP virus (VII+) or 293 cells expressing Cre recombinase (VII–) infected with the Ad5-VII-loxP virus. Virus particles from the Ad5-VII-loxP-infected cells were harvested 48 and 72 hr post-infection (hpi). Protein molecular weight markers are indicated on the left. Major Ad capsid proteins are indicated on the right. (D) Western blot analyses of purified virus particles described in (C). Ad capsid proteins are indicated on the right. (E) Western blot analysis of purified virus particles described in (C), plus ts1 particles isolated following infection of cells at the restrictive temperature, probed with antibodies directed against pVI/VI, or C-terminal peptides of pVI (VI-C) or pVIII (VIII-C). The asterisk (*) indicates full-length, unprocessed pre-VI, the bullet (•) indicates iVI, and the open circle (o) indicates fully processed VI.
Fig 3.
Infection of cells with virus particles that lack protein VII.
A549 cells (A) or HeLa cells (B) on glass coverslips were infected with 200 particles/cell wild-type Ad5 or the Ad5-VII-loxP virus grown in 293 cells (Ad5-VII+), or 2000 particles/cell VII−virus (Ad5-VII–). At 24 hours post-infection, cells were processed for IF using antibodies directed against protein VII (FITC secondary antibody; left column) and Ad DNA binding protein (TRITC secondary antibody; center column). Merged images are shown in the right column. The white bar represents 10μM.
Fig 4.
Core protein VII is required for efficient viral early gene expression and DNA replication.
(A) HeLa cells were infected with wild-type Ad5 (WT) and the VII+ and VII−viruses, and virus infection (2 hpi) and viral DNA replication (24 hpi) was quantified by qPCR. Primer pairs for qPCR hybridized to all three viruses (Total vDNA) or were specific to the pVII open reading frame (VII+ vDNA). (B) HeLa cells were infected with wild-type Ad5 (WT) and the VII+ and VII−viruses and E1a mRNA levels were quantified by RT-qPCR at 2 and 5.5 hr post-infection (hpi). The values were normalized to cellular GAPDH mRNA levels as described in Materials and methods. (C) The VII+ and VII−viruses were used to infect 293 cells and E2a, E2b, and E4 mRNA levels were quantified by RT-qPCR at 5.5 hpi and normalized to cellular GAPDH mRNA levels. Values are plotted as mean ± sd.
Fig 5.
Transfected VII– virion DNA fosters productive infection.
HeLa cells were cotransfected with purified viral DNAs and an EGFP expression vector. Viral DNAs corresponded to Ad5-WT, Ad5-VII-loxP grown in 293 cells (VII-lox-293), and two independent preparations of Ad5-VII-loxP grown in 293-Cre cells (VII-lox-Cre1 and VII-lox-Cre2). 48 hours after transfection, the cells were harvested and used for the preparation of whole cells protein extracts, total cell DNA, and total cell RNA, as described in Materials and Methods. (A) EGFP Western blot of samples isolated from three independent transfection experiments. Lane 1, untransfected; lanes 2, 6, and 10, cells transfected with Ad5-WT DNA; lanes 3, 7, and 11, cells transfected with VII+ DNA; lanes 4, 8, and 12, cells transfected with VII−DNA preparation 1; lanes 5, 9, and 13, cells transfected with VII−DNA preparation 2. (B) Total cellular DNA isolated from these transfections were digested with DpnI to cut transfected, input plasmid DNA, and analyzed by qPCR for Ad5 DNA levels using a primer pair that PCRs across a segment with two DpnI sites. (C) Total cellular RNA was these transfections was analyzed for E1a mRNA levels as described in Fig 5B. n = 3 for (B) and (C) and the values are plotted as mean ± sd.
Fig 6.
Subcellular localization of VII−virus particles analyzed by confocal microscopy.
A549 cells were infected with EdC-labeled (see Supplemental Information) Ad5-WT at 4°C for 1 hour (A, surface virus) or with Ad5-WT (B, HAdV-C5_wt) or VII−virus particles (C, HAdV-C5_ΔVII) at 37°C for 30 min. Cells were subsequently incubated for 0 min (30min + 0min), 3 hours (30min + 180min), or 5 hours (30min + 300min). Cells were processed for IF using antibodies directed against intact or partly fragmented Ad5 capsids (hexon, left column, white dots) followed by Click reactions to crosslink Alexa Fluor 488-azide to EdC-labeled viral DNA (center column, DNA, white dots). Panels in the right column represent merged, color images including nuclear DAPI staining. The white bar represents 10μM.
Fig 7.
Subcellular localization of VII−virus particles analyzed by transmission electron microscopy.
A549 cells were infected with VII+ or VII−virus particles at 37°C for 60 min, washed in warm virus-free medium, further incubated at 37°C for 5 or 240 min, fixed in glutaraldehyde, and processed for Epon embedding and thin section EM analysis. (A) Representative images of virions in the cytosol or the nuclear membrane (arrows), and in vesicles (arrowheads). (B) Quantification of virions at the plasma membrane (blue bars), in vesicles (red), the cytosol (green), and on the nuclear membrane (violet). The Top panel represents the raw data, including the number of virions (v) and cells (c) for each condition. The Bottom panel shows the normalized data as a fraction of the total number of virions for each condition. Note the enrichment of VII-less virions in multi-vesicular structures, and the strong reduction in wild-type virions 300 min post-infection, indicative of capsid disassembly [49, 88, 89]. The black bar in (A) represents 0.1μm.