Representative projections of the entire volume of PBCV-1 infected cells.

<p>A, B. Views of the entire volume of de-convoluted fluorescence images showing 3h PI PBCV-1 infected cells stained for DNA (Sytox; green) capsids (anti-capsid antibody, magenta) and chloroplast auto-florescence (red). Arrows point to the possible infecting viruses near the chloroplasts. New viral progeny packed with DNA are represented by green dots in the cytoplasm of the cells, indicating successful infection. Scale bars: 2 μm.</p

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting <i>Paramecium bursaria</i> chlorella virus-1

<div><p>A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA <i>Paramecium bursaria</i> chlorella virus-1 <i>(</i>PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.</p></div

Host and viral membrane fusion generates a tunnel though which viral DNA is ejected.

<p>A-G. PBCV-1-infected chlorella cells at 1.5–2 min PI were immobilized with HPF-FS and thick sections were analyzed by STEM tomography. A. A 5.2 nm tomographic slice from a 220 nm-thick STEM tomogram showing the close proximity between the viral and host internal membranes resulting from their convergence at the infection site. B. A 7.8 nm tomographic slice of a high magnification of the inset in panel A. C. A 5.2 nm tomographic slice from a different 220 nm STEM tomogram. D. High magnification of the inset of panel C. The generation of a continuous tunnel is evident. E, F. Two different 5.2 nm STEM tomography slices from the same tomogram showing the same PBCV-1-infected cells with almost completely empty capsids in which the membrane tunnel is still detected. G. A 5.2 nm tomographic slice from a 216 nm-thick STEM tomogram exhibiting an empty capsid attached near thylakoid membrane stacks (red arrowheads). In all panels the membrane tunnel and the protrusion of the host membrane are marked with blue and white arrowheads, respectively. Asterisk: viral DNA. H, I. Volume rendering representation of the STEM tomogram shown in panel A. The 3D surface representation highlights the barriers that viral DNA has to overcome to reach the host nucleus (including cell wall, plasma membrane, cytoplasmic vesicles, Golgi, and photosynthetic membranes that were not captured in this tomogram). A PBCV-1 virion is attached to the cell wall (brown). The host membranes as well as cytoplasmic vesicles are marked in blue. The capsid is depicted in yellow, the internal viral membrane and the membrane tunnel (arrowheads) are shown in blue. Viral DNA is shown in green. Scale bars: A, C, G: 100 nm; B, D, E, F: 50 nm.</p

Primers used to determine viral DNA purity.

<p>Primers used to determine viral DNA purity.</p

PBCV-1 infected chlorella cells at 2 min PI.

<p>A, C. TEM micrographs highlighting the hurdles that are associated with trafficking large viral DNA genomes to the nucleus. The large chloroplast is visible with an adjacent virus attached. The nucleus is located at the opposite side of the cell. B, D. High magnifications of panels A and C, respectively, showing the tightly packed thylakoid membrane (red arrowheads) and PBCV-1 virions attached adjacent to the thylakoid membrane stacks. Note that Panel D is rotated by 90o counter clockwise relative to Panel C. Scale bars: A, C: 500 nm; B, D: 100 nm.</p

A model depicting early stages of PBCV-1 infection and highlighting the similarity between PBCV-1 infection and that of bacteriophages.

<p>A. The main components involved in PBCV-1 infection, including the PBCV-1 icosahedral capsid (yellow), internal membrane (dark blue), genome (gray) and spike (magenta) on the left side of the panel, as well as the cell wall (brown), cellular membrane (dark blue), thylakoid stacks (green), nuclear membrane (light blue) and nucleus (gray) of the host on the right side. B-D. Spike-mediated perforation of the host cell wall, protrusions of the viral and cellular internal membranes and their subsequent fusion into a membrane tunnel. D, E. Internalization of PBCV-1 genome into the host cytoplasm through a membrane tunnel, accompanied by the perforation of the photosynthetic thylakoid stacks and apparently promoted by viral DNA condensation. Significantly, perforation of the host cell wall, generation of a membrane portal and genome condensation represent crucial infection stages of bacteriophages (see text for details).</p

DNA immunolabling and in-situ hybridization of mock-infected chlorella cells.

<p>A, B. Mock-infected cells were cryo-immobilized and thin sections were immuno-labeled with anti-DNA antibodies. A. Low magnification view of a mock-infected chlorella cell. B. High magnification view of the cell shown in panel A. Whereas the nucleus exhibits dense DNA labeling, no cytoplasmic DNA labeling is detected. C, D. Mock-infected cells were processed for EMISH and thin sections were labeled with PBCV-1 specific DNA probes. C. Low magnification view of a mock-infected chlorella cell. D. High magnification view of the nucleus region revealing no DNA labeling, thus further validating the specificity of the viral DNA probes. Nu: nucleus. Scale bars: A, B, C: 500 nm; D: 200 nm.</p

STORM analyses of PBCV-1 infected cells reveal condensed cytoplasmic viral DNA.

<p>Cells were infected for 1.5–2 min and processed for immuno-florescence with anti-PBCV-1 capsid antibodies, counterstained with SYTOX Orange for DNA detection and analyzed by STORM. A. A STORM image showing a host cell with two capsids at its periphery (yellow). Inset shows a virus delivering its genome (green) into the host cytoplasm at the opposite side of the nucleus. B. Another chlorella cell with an adjacent virus particle in the process of delivering its DNA into the nucleus (white arrowhead). In both panels the dashed line marks the nuclear boundary. Nu: nucleus. Scale bars: 1 mm.</p

PBCV-1 genomes are transported towards the host nucleus.

<p>A-D. Cells infected with PBCV-1 for 2–6 min, immobilized using HPF-FS and thin sections were immuno-labeled with anti-DNA antibodies. A. Low magnification view of an infected cell. B. High magnification of the region indicated in panel A revealing a partially empty capsid (arrow) in the process of DNA ejection and translocation to the nucleus (arrowhead; nucleus contour is delineated with a dashed white line). Note that the viral DNA bypasses cytoplasmic barriers. C. Low magnification of a cell that was infected with PBCV-1 exhibiting dense DNA labeling close to the nucleus. D. High magnification of the cell in panel C. The dense and clustered DNA labeling near the nucleus strongly implies a condensed viral DNA morphology. Inset shows high magnification of the cytoplasmic DNA (nucleus borders are delineated with a dashed line). E, F. Cells were infected with PBCV-1 for 6 min and then chemically fixed and thin sections were subjected to EMISH. E. Low magnification of a cell illustrating dense viral DNA near the nucleus. F. High magnification view of panel E. Note that viral DNA is on a possible entry into the nucleus (white arrowheads in the inset). Nucleus contour is delineated with white dashed line. Nu: nucleus, Ch: chloroplast. Scale bars: A, C, D, E: 500 nm; B, F: 200 nm.</p