ORF7 of varicella-zoster virus is a neurotropic factor

Abstract

Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens.

Fig 1

ORF7 is required for VZV replication in human neurons in vitro. (A) ARPE-19 and SH-SY5Y cells were plated on glass coverslips in 6-well tissue culture plates and infected with 100 PFU of cell-free VZV particles (MOI, 8.3e−5). SH-SY5Y cells were fully differentiated at the time of infection. At 7 dpi, viral plaques were detected by EGFP expression (green) in WT-, 7D-, and 7R-infected ARPE-19 cells, as well as in WT- and 7R-infected SH-SY5Y cells. Although some isolated EGFP-positive cells are seen in 7D-infected SH-SY5Y cells, no plaques were detected 7 dpi. (B) hESC-derived neuronal cells, prepared as described in Materials and Methods, were infected with cell-free WT and 7D VZV, and live cultures were visualized at 2 and 7 dpi. Green-fluorescent plaques were detected in WT but not in 7D infections at the experimental endpoint, despite the presence of sparse EGFP-positive cells. Right panels in each set represent merged visible and fluorescence images of the left panels. (C) ARPE-19 and SH-SY5Y cells seeded on glass coverslips in 6-well plates were infected with 100 PFU of WT, v-Oka, 7D, and 7R cell-free particles and then fixed and probed with mouse monoclonal antibodies against the viral envelope glycoprotein gE (red, upper panels). Nuclei were detected using DAPI (blue) in the merged lower panels. Plaques were visualized at 7 dpi. Widespread expression of gE in plaque patterns indicates productive viral replication in all samples except for 7D-infected SH-SY5Y cells, where only a few isolated cells stain positive for gE. Notably, WT, 7R, and v-Oka showed robust proliferation in SH-SY5Y cells.

Fig 2

ORF7 is required for syncytium formation. (A) ARPE-19 cells were infected with 100 PFU of cell-free WT, 7R, or 7D (MOI, 8.3e−5) and visualized at 7 dpi. Plaque sizes were averaged from a minimum of 40 plaques per virus. Error bars represent standard errors. Notably, the 7D mean plaque size was about 16% of that of WT, while WT and 7R plaque sizes were similar. (B) ARPE-19 cells were seeded in 6-well plates and infected with 200 PFU of WT, 7D, or 7R cell-free VZV or mock infected (PBS). Viral replication was measured daily for 8 days by adding d-luciferin to the wells and recording the bioluminescence signal with the IVIS50 imaging system. The total photon count (photons/s/cm²/steradian) was averaged from 3 samples per virus. Error bars represent standard deviations. The growth kinetic difference between 7D and WT was statistically significant (P < 3.95e−09, ANOVA); however, the presence of 7D plaques suggests that ARPE-19 cells have a compensatory mechanism allowing viral spread in the absence of ORF7. (C) Live ARPE-19 cells infected with WT (upper panels) or 7D (lower panels) cell-free virus and visualized 7 dpi. Left panels show live images of green-fluorescent viral plaques stained with cell membrane WGA-Alexa Fluor 594 probe (red). Middle panels show cell nuclei stained with DAPI (blue) and cell membrane probe (red). Right panels are merged images of left and middle panels. The white arrows indicate multinucleated cells (syncytia) in the WT infection, but no indication of syncytia is seen in 7D. Similar results were obtained using cell-associated viruses.

Fig 3

ORF7 is a viral component that localizes to the Golgi network in infected cells. (A) Cell lysates from WT- and 7D-infected cells were subjected to a Western blot assay and probed with mouse monoclonal antibody against ORF7 and HRP-conjugated goat anti-mouse secondary antibodies. Probing with anti-gE antibody (left) served as a positive control. One distinct band of the anticipated size (29 kDa) corresponding to ORF7 product was detected in the WT sample (right). (B) WT cell-free purified particles (V) and WT-infected cell lysates (C) were subjected to a Western blot assay using anti-ORF7 (panel 1), anti-gE (panel 2), anti-EGFP (panel 3), and antiactin (panel 4) mouse monoclonal antibodies. As expected, gE, being a viral envelope constituent, was present in both samples, whereas EGFP and actin were present only in the cell lysates, since they are not packaged into the virion. ORF7 was detected in the cell-free purified VZV, indicating that it is likely an integral component of the virion, as predicted. (C) In order to determine subcellular localization, GFP-negative WT-infected ARPE-19 cells were stained with mouse anti-ORF7 (green). ORF7 was detected in a discrete punctate pattern in the perinuclear region. The Golgi network was labeled with rabbit antigiantin antibodies (red, left), and mitochondria were labeled with human antimitochondrial antibodies (red, right). Examples of infected (I) and uninfected (U) cells are shown with white arrows. Nuclei were visualized using Hoechst 33258 (blue). These images indicate that ORF7 colocalizes with the Golgi network but not with the mitochondria within infected cells. (D) Mock infected ARPE-19 cells (left), as well as WT-infected ARPE-19 cells (middle) and SH-SY5Y cells (right), were fixed and stained with anti-ORF7 antibodies (red). ORF7 has similar distributions in the two cell types.

Fig 4

ORF7 is essential for viral replication in human neurons ex vivo and in vivo. (A) Human fetal DRG cultured on NetWell inserts were infected with cell-free WT and 7D viruses at 100 PFU/well. Over a period of 20 days, the bioluminescence signal was recorded every 48 h with the IVIS system after adding d-luciferin to the culture medium. The growth curve generated represents total photon counts (photons/s/cm²/steradian) averaged from 4 samples per virus, and the error bars represent the standard deviations. (B) Human fetal DRG implanted under the kidney capsule of SCID mice were infected by surgical exposure and direct injection of DRG with 100 PFU of cell-free WT, 7R, and 7D VZV, respectively. Luciferase activity in the infected tissue was detected every other day after administration of d-luciferin intraperitoneally for 10 dpi. The growth curve was generated from 5 mice per virus as described for panel A. (C) At 10 dpi, WT- and 7D-infected DRG were harvested from SCID mice and fixed. Sections were stained for gE (brown) and counterstained with hematoxylin (blue). Lower panels represent higher-magnification pictures of selected areas of the upper panels. Red arrowheads indicate neurons. Vacuolization and possible neuron-satellite cell aggregates (black arrows) were observed in the WT-infected sample, correlating with productive replication and widespread presence of viral gE antigen (brown), while 7D samples retained normal DRG morphology and no gE was detected. (D) FISH staining of the samples described in panel A was performed using VZV genomic DNA probes (green). In WT-infected tissue (left panels), intranuclear signal was detected from nuclei of both neurons (red arrowheads) and adjacent satellite cells (white arrowheads), indicative of active viral DNA replication within intranuclear replication centers. In contrast, 7D DNA was not detected in any of the samples tested at the experimental endpoint.

Fig 5

TEM analysis of infected DRG xenografts shows no evidence of virion production in the 7D samples. TEM images taken from 7D (A to C)- and WT (D to F)-infected DRG samples. The nuclei of sensory neurons (N) and surrounding satellite cells (S) are labeled. In the 7D sample, no evidence of intranuclear capsids (B; box I in panel A) or of cytoplasmic virions (C; box II in panel A) and no tissue damage were observed, whereas the WT-infected DRG sample shows some vacuolization in the plasma membrane region (D), accompanied by the presence of polymorphic virions (white arrows) in the cytoplasm of the infected neuron (E; box I in panel D), as well as intranuclear capsids (F; box II in panel D).