The replication cycle of varicella-zoster virus: analysis of the kinetics of viral protein expression, genome synthesis, and virion assembly at the single-cell level

Abstract

Varicella-zoster virus (VZV) is a human alphaherpesvirus that is highly cell associated in cell culture. Because cell-free virus yields are too low to permit the synchronous infections needed for time-resolved analyses, information is lacking about the sequence of events during the VZV replication cycle. To address this challenge, we differentially labeled VZV-infected inoculum cells (input) and uninfected (output) cells with fluorescent cell dyes or endocytosed nanogold particles and evaluated newly infected cells by confocal immunofluorescence or electron microscopy (EM) at the single-cell level at defined intervals. We demonstrated the spatiotemporal expression of six major VZV proteins, ORF61, IE62, IE63, ORF29, ORF23, and gE, representing all putative kinetic classes, for the first time. Newly synthesized ORF61, as well as IE62, the major VZV transactivator, appeared within 1 h, and they were targeted to different subnuclear compartments. The formation of VZV DNA replication compartments started between 4 and 6 h, involved recruitment of ORF29 to putative IE62 prereplication sites, and resulted in large globular nuclear compartments where newly synthesized viral DNA accumulated. Although considered a late protein, gE accumulated in the Golgi compartment at as early as 4 h. ORF23 capsid protein was present at 9 h. The assembly of viral nucleocapsids and mature enveloped VZ virions was detected by 9 to 12 h by time-resolved EM. Although syncytium formation is a hallmark of VZV infection, infection of neighboring cells did not require cell-cell fusion; its occurrence from 9 h is likely to amplify VZV replication. Our results define the productive cycle of VZV infection in a single cell as occurring in 9 to 12 h.

FIG. 1.

Differentiation of cells newly infected with VZV from the infected-cell inoculum. The labeling method and selective analysis of newly infected (output) cells are outlined in the upper left panel. Examples of the spatiotemporal expression patterns of several viral proteins in newly infected cells (white stars) are shown. Unlabeled and uninfected (output) HELF cells were seeded on coverslips and infected with green-CellTracker-labeled and infected inoculum cells. The cells were then fixed at various time points and stained with specific antibodies for VZV proteins (red) IE62 and ORF61 (fixation at 4 h), IE63 and ORF29 (fixation at 6 h), or gE and ORF23 (fixation at 9 h) and Texas Red-conjugated secondary antibodies. Viral DNA (vDNA; red) was detected by DNA in situ hybridization (fixation at 6 h). The cell nuclei (blue) were counterstained with Hoechst 22358. The white arrow in the panel for ORF23 marks a newly infected cell nucleus that is also shown at higher magnification (inset). Scale bars are 20 μm.

FIG. 2.

The patterns of spatiotemporal expression of selected VZV immediate-early, early, and late proteins and viral genomic DNA. Fixation for this time course analysis was at 0 h (not shown), 2 h, 4 h, 6 h, 9 h, and 12 h after inoculation of unlabeled HELF cells with input cells labeled with green CellTracker. The cells were then stained with specific antibodies for VZV proteins (red, from left to right) ORF61, IE62, IE63, ORF29, gE, and ORF23 followed by Texas Red-conjugated secondary antibodies. VZV DNA was detected by DNA in situ hybridization (red). Cell nuclei were counterstained with Hoechst 22358. Ten fields of 30 to 50 output cells were scanned to determine the representative staining patterns in newly infected cells at each time point. Single representative cells or nuclei are shown. PM, plasma membrane; cytoplas., cytoplasm.

FIG. 3.

ORF61 and IE62 proteins are targeted to different subnuclear compartments and require active transcription and translation but not viral DNA synthesis for very early expression. (A) Unlabeled HELF cells were infected and fixed after 60 min (left panels) or 90 min (right panels). The cells were then double stained with either a combination of rabbit polyclonal anti-ORF61 antibody (upper panels, red) and anti-PML (blue; monoclonal mouse) or with rabbit polyclonal anti-IE62 (lower panels, red) and anti-PML (blue; monoclonal mouse). Secondary antibodies were donkey anti-rabbit Alexa Fluor 546 and donkey anti-mouse Alexa Fluor 647. Uninfected control cells (insets) were stained with the same antibody combinations. The small images in the ORF61 panels show single-PML (blue)- or ORF61 (red)-staining patterns that completely overlap (pink) in the larger overlay images. The two images shown in the IE62 panels (at 60 min or 90 min) depict the same nucleus. In the images on the left, nuclear staining (Hoechst 22358) is shown with the IE62 (red) and PML (blue) signals to clearly mark the nuclear areas. In the images on the right, the nuclear staining was left away for better visibility of the IE62 punctae (red; white arrows) that do not overlap with the PML signal (blue). Scale bars are 10 μm. (B) Uninfected HELF cells were pretreated for 30 min with cycloheximide, actinomycin D, or PAA or left untreated (control) and then inoculated with infected, untreated, green-labeled input cells. Incubation occurred in the presence of the same drugs and was stopped by fixation at 4 h. Cells were then stained for either ORF61 (red, upper panels) or IE62 (red, lower panels), and nuclei (blue) were counterstained with Hoechst 22358. Output cells adjacent to infected inoculum cells (green cytoplasmic staining plus red nuclear staining of ORF61 or IE62) were analyzed for nuclear expression of ORF61 or IE62 (red). Nuclear ORF61 or IE62 (red) is visible in several newly infected cells (white stars) in either the control (far left panels) or the PAA-treated cells (far right panels) but not in the cycloheximide- or actinomycin D-treated cells (middle panels). Scale bars are 20 μm.

FIG. 4.

The single-stranded DNA binding protein ORF29 is targeted to IE62 nuclear domains that transform into viral replication compartments. (A) HELF cells were seeded on glass coverslips and infected with green-labeled inoculum cells for 4 h, 6 h, or 8 h and were double stained with anti-IE62 (green; mouse monoclonal antibody) and anti-ORF29 (red; polyclonal rabbit antibody) followed by secondary anti-mouse Alexa Fluor 488 or anti-rabbit Texas Red-conjugated secondary antibodies. Nuclei (upper panels a through d; blue) were counterstained with Hoechst 22358. Newly infected cells were identified by the absence of green cytoplasmic staining and were analyzed for IE62 and ORF29 expression by confocal microscopy. The upper panels (a through d) show an overview of the nuclei with merged blue, green, and red channels. Areas of the same nuclei (within the white squares in the upper panels) are shown at higher magnification in lower panels a' through d' (for better visualization of red and green signals, no blue channels are shown). White arrows point to red ORF29 punctae associated with green-stained IE62 nuclear domains. Scale bars are 10 μm. (B) HELF cells were seeded and infected and then additionally treated at 12 h after infection with a 30-min pulse of 0.1 mM BrdU (in medium) before being fixed at 12.5 h. Cells were then stained with anti-ORF29 (red) or -ORF62 (green; polyclonal antibody) and Texas Red (ORF29)- or FITC (IE62)-conjugated secondary antibodies, respectively, processed for DNA in situ hybridization (red; DIG-labeled VZV DNA probe), or stained for incorporated BrdU (red; biotinylated anti-BrdU monoclonal antibody and streptavidin-Texas Red conjugate for secondary detection). Scale bars are 10 μm. Each of these single stainings reveals similarly shaped globular nuclear compartments. (C) Cells from the same experiment as for panel B were double stained for either BrdU (a, red) with viral DNA (vDNA; b, green), BrdU (d, red) with ORF29 (e, green), or ORF29 (g, red) with IE62 (h, green). The merged channels are shown in panels c, f, and i. Control stainings for ORF29, viral DNA, and BrdU are shown in panels j, k, and l. Scale bars are 10 μm.

FIG. 5.

VZV nucleocapsids and surface expression of glycoprotein gE are detectable at 9 h after infection. (A) Uninfected HELF cells were inoculated with green-labeled inoculum cells and fixed after 9 h. Unpermeabilized cells were stained with anti-gE (left panel, red; monoclonal antibody) and a secondary anti-mouse Texas Red antibody. White arrows mark areas of plasma membrane-expressed gE protein (red). Other cells were permeabilized and stained with rabbit polyclonal anti-ORF23 (right panel, red) and a secondary anti-rabbit Texas Red-conjugated antibody. Newly infected cells are marked with a white star. Scale bars are 30 μm. (B) Time-resolved standard EM analysis of newly infected HELF cells. (a) Overview of an infected HELF cell 9 h after inoculation. The Golgi-compartment area (b, white square) is shown at higher magnification in the inset panel b', which shows a nucleocapsid (arrow) undergoing envelopment with a Golgi-compartment-derived membrane. The white arrows labeled c through f in the overview image point to structures that are shown at much higher magnification in the images on the right (c through f). (c and d) Nuclear viral nucleocapsids are indicated by arrows. (e and f) Lysosomes with clusters of internalized 10-nm gold particles (arrows) that were used to discriminate newly infected cells from infected inoculum cells are shown.

FIG. 6.

Demonstration of cell-cell fusion events at 9 h after VZV infection. HELF cells were seeded on glass coverslips and stained with orange CellTracker (CMRA), washed, and then inoculated with green-CellTracker-labeled uninfected HELF cells (a through c) or infected HELF cells (d through l). Cells were fixed at 4 h (d through f) or 9 h (g through i and j through l) after infection and stained for gE protein (blue; monoclonal antibody and secondary anti-mouse Alexa Fluor 647 conjugate). Nuclei were stained with Hoechst 22358 (d through l, gray). Cells were analyzed for the mixing of green and red fluorescent signals in the cytoplasm that would indicate fusion of newly infected cells with an input cell. The thin white arrows in panels g through i point to a cell (red and green cytoplasm) that seems to have partially fused with fused input cells. The adjacent output cell (arrowhead) has not yet fused (only red fluorescence). Scale bars are 50 μm.

FIG. 7.

Detection of VZV nucleocapsids, intracellular enveloped virions in cytoplasmic vacuoles, and extracellular enveloped virions at 12 h after infection. Areas of two cells (A through F, cell 1; G through I, cell 2) that were fixed after 12 h of infection are shown at the ultrastructural level after processing for time-resolved standard EM. (A) Overview of cell 1 with black squares indicating the areas that are shown in panels B through D at a higher magnification. (B) Demonstration of many nucleocapsids (arrows) in the nucleus. (C) Stack of Golgi cisternae (G) with adjacent nucleocapsids (small arrows) and one enveloped nucleocapsid (arrowhead). The inset shows extracellular virions associated with the plasma membrane of cell 1. (D and E) Another Golgi stack (G) in the same cells with modified Golgi cisternae (D, arrows) and enrichment of electron-dense putative tegument proteins (E, arrowheads) at the concave sides of these cisternae. (F) A multilamellar lysosome (L) is shown with a cluster of internalized 10-nm gold particles that was used to identify cell 1 as a newly infected cell (see Materials and Methods). (G) Overview of cell 2. The black square marks an area shown at higher magnification in panel H. (H) A nucleus with nucleocapsids (left side) is visible, and three cytoplasmic vacuoles (v) that contain VZV particles (arrows) can be seen. An extracellular virion is also marked (right side, arrow). (I) Three lysosomes are marked (L), one of which harbors a cluster of 10-nm gold particles (white square) that are clearly visible at a higher magnification (arrow in inset).

FIG. 8.

Model of the cell-to-cell spread of VZV before cell fusion in vitro. Mature VZV particles are transported in an export vacuole to the plasma membrane of an infected inoculum cell (gray cell on left). (A) VZV particles released by exocytosis adhere to the plasma membrane at the interface with culture media. Infectious VZV particles are not released into the media or rapidly lose infectivity, since secondary plaques do not form in VZV-infected monolayers. (B) Some VZV particles are released from the inoculum cell in close proximity to the plasma membrane of the adjacent uninfected cell (white cell on right). (C) Envelope glycoproteins of extracellular VZV particles at these sites have an increased probability of binding to cell surface receptors on the plasma membrane of the uninfected cell. Entry into the adjacent cell occurs by endocytosis (D) or direct entry (E), which is followed by the release of partially tegumented capsids into the cytoplasm of the newly infected cell. Our model predicts that the spread of cell-associated extracelluar VZ virions in vitro is determined by the proximity of infected and uninfected cells and does not require cell fusion.