Fig 1.
HAdV5 tolerates fusion of proteins pV and pIX to mCherry.
(A) Overview of mCherry-fusion constructs. Two HAdV5 mutants were generated. Protein V and protein IX were fused to the mCherry fluorophore in combination with a varying flexible linker region. mCherry was fused N-terminally to pV and C-terminally to pIX without disrupting adenoviral splice sites. (B) Restriction analysis of wt, pV-mCherry and pIX-mCherry bacmid DNA (lanes labelled A, B, C) including in-silico-prediction of restriction pattern. (C) negative stain TEM of purified virus particles. The virus particles of each mutant were purified, contrasted with uranyl acetate negative stain and imaged by transmission electron microscopy. Scalebars indicate 100 nm. HAdV5 pV-mCherry particles are shown in i) HAdV5 pIX-mCherry particles are shown in ii). (D) Growth curve of virus particle release from infected A549 cells from 0 to 96 hpi.
Fig 2.
pV-mCherry and pIX-mCherry display a large intranuclear compartment late in infection.
(A) Infection of A549 cells with HAdV5 pV-mCherry at 24 hpi and 48 hpi. (B) Infection of A549 cells with HAdV5 pIX-mCherry at 24 hpi and 48 hpi. The cells were imaged by live-cell confocal laser-scanning fluorescence microscopy. A representative cell is shown for each condition. The dsDNA signal is represented by Hoechst 33342 stain (Hoechst). The nuclear lamina is represented by a GFP-nanobody recognizing lamin A (Lamin A). pV and pIX localization is detected through the viral pV-mCherry and pIX-mCherry fusion construct (pV-mCherry/pIX-mCherry). The signal overlap is represented in color (merge). Nuclear regions of interest are enlarged (zoom) with colored corners indicating the channel color. Scalebars indicate 10 μm.
Fig 3.
Antibodies against pV and pIX do not fully stain the LVAC.
(A) Immunofluorescence labeling of pV in HAdV5 wt infection. (B) Immunofluorescence labelling of pIX in HAdV5 wt infection. A549 cells were infected with HAdV5 wt, fixed at 48 hpi and imaged by confocal laser-scanning fluorescence microscopy. Cells were stained with Hoechst 33342 (Hoechst), and immunostained against pV (anti-pV) or pIX (anti-pIX) and DBP (anti-DBP). The nuclear lamina is represented by a GFP-nanobody recognizing lamin A (Lamin A). The signal overlap is represented in color (merge). A representative non-infected and infected cell is shown for each stain. Scalebars indicate 10 μm.
Fig 4.
The signal of mCherry fusion constructs and pV and pIX antibody only diverges in late infection.
(A/C) Immunofluorescence stain of pV-mCherry/pIX-mCherry with anti-pV/pIX antibody. A549 cells were infected with HAdV5 pV-mCherry and HAdV5 pIX-mCherry. At 24 and 48 hpi the cells were fixed and immunostained with pV or pIX antibodies and imaged in confocal fluorescence microscopy. A representative cell for both time points of HAdV5 pV-mCherry and HAdV5 pIX-mCherry infection is shown. Scalebars indicate 10 μm. (B/D) Quantification of mCherry-antibody signal colocalization as represented by the average Pearson’s coefficient r and Mander’s coefficients M1 and M2 (n = 12). Statistical significance was calculated using Student’s t-test with preceding F-test and is indicated as stars above the bars.
Fig 5.
pV forms ring-like structures within the nucleus before congregating into the LVAC.
(A) Infection progression of HAdV5 pV-mCherry infected cell. (B) Infection progression of HAdV5 pIX-mCherry infected cell. The cells were selected for imaging in live-cell spinning-disk confocal fluorescence microscopy at 24 hpi. The initial nuclear boundaries for the HAdV5 pV-mCherry or pIX-mCherry infected cell are visualized as a dashed line. The cells were imaged as z-stack to be able to adjust for z-movement of infected cells. The image histograms along infection progression have been modified to adjust for changes in protein intensity. (C) Co-stain of pV showing the ring-like phenotype and DBP replication centers in HAdV5 wt infection. A549 cells were infected with HAdV5 wt, fixed at 24 hpi, stained with Hoechst 33342 (Hoechst), and immunostained against pV (anti-pV) and DBP (anti-DBP). Cells were imaged by confocal laser-scanning fluorescence microscopy. The signal overlap is represented in color (merge). A DBP replication ring is enlarged (zoom) with colored corners indicating the channel color. Scalebars indicate 10 μm.
Fig 6.
Inside the LVAC, pIX-spots are located within cavities of a dsDNA and pV honeycomb-like organization.
(A) Colocalization of pV-mCherry and pIX-mCherry and dsDNA in the LVAC. A549 cells were infected with HAdV5 pV-mCherry and HAdV5 pIX-mCherry. At 48 hpi, the cells were stained with Hoechst 33342 and imaged in confocal laser-scanning live-cell fluorescence microscopy. A representative cell for each virus mutant is shown. (B) Colocalization quantification between Hoechst 33342 and pV-mCherry or pIX-mCherry signal in the LVAC. The overlap between both signals is represented by the average Pearson’s coefficient r and Mander’s coefficients M1 and M2 (n = 12). Statistical significance was calculated using Student’s t-test with preceding F-test and is indicated as stars above the bars. C) Colocalization of pV-mCherry and pIX-mNeongreen in the LVAC. A549 cells were infected with the double mutant HAdV5 pV-mCherry pIX-mNeongreen and imaged at 48 hpi. A region of the LVAC is enlarged (zoom) showing pIX-mNeongreen spots to populate the cavities of the pV-mCherry honeycomb. Colored corners indicate the channel color. Scalebars indicate 10 μm.
Fig 7.
Paracrystalline virus arrays accumulate within the LVAC and correspond to the pIX-mCherry spots found in the LVAC.
(A) A549 cells infected with HAdV5 pV-mCherry. (B) A549 cells infected with HAdV5 pIX-mCherry. The cells were stained with Hoechst 33342 and were imaged by live-cell spinning-disk confocal fluorescence microscopy at 48 hpi (FM). Immediately after imaging, the cells were fixed and prepared for transmission electron microscopy (TEM). 50 nm sections of the epon-embedded cells were imaged with TEM. FM cell overviews are shown in images i) with Hoechst 33342 in blue and pV-mCherry and pIX-mCherry in red. TEM cell overviews are shown in images ii) including scalebars indicating 5 μm. Higher magnification images of selected areas of cells are shown in images iii) and iv) for both sections A) and B) including scalebars indicating 0.5 μm. Scalebars for fluorescence images indicate 10 μm.
Fig 8.
The paracrystalline arrays are highly immobile within LVAC.
(A) FRAP laser bleaching of pV-mCherry and pIX-mCherry signal within LVAC constituents in infected A549 cells. A dashed red circle is indicating the bleach area. The cells are shown before bleaching (pre-bleach), at the timepoint of bleaching (bleach) and after 2 min recovery (2 min post-bleach). (B) FRAP quantification. The relative fluorescence intensity change was plotted against time. The average signal change is indicated (n = 6) with the standard deviation indicated as colored underlying area. (C/D) Time-lapse live-cell fluorescence microscopy analysis of A549 cells infected with pIX-mCherry. The position of paracrystalline virus arrays (pIX-mCherry spots) remained largely unchanged over 54 min (indicated by white arrows) and only slowly shifted in the course of multiple hours. Images in D) were denoised using the Noise2Void algorithm [29]. Scalebars indicate 10 μm.
Fig 9.
Model of late virion accumulation compartment (LVAC) formation and localization of viral proteins within.
The nucleus is represented as a grey ellipse. At 24 hpi, pV is located around DBP replication centers in areas accumulating replicated genomes. At this timepoint, pIX was not found in the nucleus but was instead localized in the cytoplasm (not displayed in model). During transition between 24 and 48 hpi the viral proteins congregated to form the LVAC. Within the LVAC, pIX spots indicate the areas of accumulated assembled virus capsids in areas neighboring viral genomes and pV. Each protein localization is accompanied by a fluorescence microscopy image of the protein observed in our study.
Table 1.
List of primers used for PCR amplification of ccdB-amp cassette.