ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells

Abstract

Although a varicella-zoster virus (VZV) vaccine has been used for many years, the neuropathy caused by VZV infection is still a major health concern. Open reading frame 7 (ORF7) of VZV has been recognized as a neurotropic gene in vivo, but its neurovirulent role remains unclear. In the present study, we investigated the effect of ORF7 deletion on VZV replication cycle at virus entry, genome replication, gene expression, capsid assembly and cytoplasmic envelopment, and transcellular transmission in differentiated neural progenitor cells (dNPCs) and neuroblastoma SH-SY5Y (dSY5Y) cells. Our results demonstrate that the ORF7 protein is a component of the tegument layer of VZV virions. Deleting ORF7 did not affect viral entry, viral genome replication, or the expression of typical viral genes but clearly impacted cytoplasmic envelopment of VZV capsids, resulting in a dramatic increase of envelope-defective particles and a decrease in intact virions. The defect was more severe in differentiated neuronal cells of dNPCs and dSY5Y. ORF7 deletion also impaired transmission of ORF7-deficient virus among the neuronal cells. These results indicate that ORF7 is required for cytoplasmic envelopment of VZV capsids, virus transmission among neuronal cells, and probably the neuropathy induced by VZV infection.IMPORTANCE The neurological damage caused by varicella-zoster virus (VZV) reactivation is commonly manifested as clinical problems. Thus, identifying viral neurovirulent genes and characterizing their functions are important for relieving VZV related neurological complications. ORF7 has been previously identified as a potential neurotropic gene, but its involvement in VZV replication is unclear. In this study, we found that ORF7 is required for VZV cytoplasmic envelopment in differentiated neuronal cells, and the envelopment deficiency caused by ORF7 deletion results in poor dissemination of VZV among neuronal cells. These findings imply that ORF7 plays a role in neuropathy, highlighting a potential strategy to develop a neurovirulence-attenuated vaccine against chickenpox and herpes zoster and providing a new target for intervention of neuropathy induced by VZV.

Keywords: ORF7; cytoplasmic envelopment; differentiated SH-SY5Y cells; differentiated neural progenitor cells; varicella-zoster virus.

FIG 1

Growth of rOka, 7D, and 7R in different cell types. (A to E) Growth curves in ARPE-19 cells (A), NPCs (B), dNPCs (C), SY5Y cells (D), and differentiated SH-SY5Y (dSY5Y) cells (E). Cultured cells (3.5 × 10⁵ cells per 35-mm dish) were infected with cell-free rOka, 7D, and 7R, respectively, at an MOI of 0.001. Images of infected cells were captured at 3 dpi are shown (left panel). Scale bar, 50 μm. Growth curves of rOka, 7D, and 7R were generated based on the total photon counts of luciferase bioluminescence. The virus replication levels were measured every 24 h by adding d-luciferin into the dishes and recording the bioluminescence signal with an in vivo fluorescence imaging system (IVIS) system. The total photon count values (photons/s/cm²/steradian) for each group at the indicated time points were collected, and the average was obtained from three independent experiments. The results are represented as averages ± the SD. Significant differences in the replication peak levels between rOka and 7D were observed in all cell groups (**, P < 0.01).

FIG 2

Transcellular transmission of VZV. (A) Construction and growth evaluation of 7D-GFP₂₃. The entire ORF7 of rOka-GFP₂₃ was replaced by kanamycin-resistant (Kan^r) gene via homologous recombination in E. coli DY380. The absence of pORF7 in 7D-GFP₂₃ was confirmed by Western blotting (left lower panel). The growth curves suggest that the growth profiles of rOka-GFP₂₃ and rOka were identical, as well as the growth curves of 7D-GFP₂₃ and 7D. (B) Virus transmission from ARPE-19 cells to dSY5Y. A diagram of the cell-seeding and virus-inoculating schema is shown (left upper panel); hydrostatic pressure was generated from the difference in medium height (higher in the left chamber). ARPE-19 cells (5 × 10⁴ cells seeded, right chamber) were infected with 5,000 PFU of rOka-GFP₂₃ (right upper panel) or 7D-GFP₂₃ (right lower panel), and virus transmission and infection signals in dSY5Y cells (2 × 10⁵ cells seeded, left chamber) were examined at 7 dpi. The green viral particles within the microchannels are indicated by dashed squares and are displayed at higher magnifications (b1 for the rOka-GFP₂₃ particle and b2 for the 7D-GFP₂₃ particle). The virus particles are indicated by the white arrows. The GFP-positive cells in both chambers were counted and are shown (left lower panel). (C) Virus transmission from dSY5Y to ARPE-19 cells. The cells were similarly seeded, the rOka-GFP₂₃ and 7D-GFP₂₃ viruses were inoculated into the left chamber, and transmissions from dSY5Y to ARPE-19 cells were examined at 7 dpi. The GFP-positive cells in both chambers were quantified and are shown.

FIG 3

Localization of ORF7 in VZV virions. (A) Localization analysis of pORF7 in purified VZV virions. Purified rOka virions were treated with trypsin in either the presence (+) or the absence (−) of Triton X-100. Equivalent amounts of cell lysates and virion samples were analyzed by Western blotting for the indicated viral proteins. (B) Localization of pORF7 in virions examined by immuno-EM. rOka- and 7D-infected ARPE-19 cells were fixed and subjected to a standard immuno-EM procedure. Images were obtained using a Hitachi H-7000FA transmission electron microscope. The colloidal gold signals of pORF7 in viral particles in the cytoplasm of infected cells are indicated by black arrows. No colloidal gold signal was observed in 7D-infected cells. The capsid protein ORF40 was also stained to clarify the location. Colloidal gold is indicated by black arrows. Scale bar, 200 nm.

FIG 4

Role of ORF7 in viral entry, genome replication, and expression of typical viral genes. ARPE-19 cells, dNPCs, and dSY5Y cells were infected with cell-free rOka, 7D, and 7R viruses, respectively, at an MOI of 0.001. The cells were harvested at 10 hpi. (A) Entry of the viruses. The harvested cells were fixed and subjected to gE IFA staining. The gE-positive cells are counted as infected cells with viral entry. (B) Viral genome replication. DNA was extracted from the cells infected with VZV and the mutants, and viral genome copy numbers were determined by qPCR. (C) Transcription of typical viral genes. Harvested cells were subjected to RNA extraction, followed by quantitation of the mRNA level of viral genes by qRT-PCR. (D) Expression of typical glycoproteins. Cell lysates were prepared from the harvested cells. Four representative glycoproteins and pORF7 were detected by Western blotting. All of the results were obtained from three independent experiments and are represented as averages ± the SD or else representative images are shown.

FIG 5

Viral particles in rOka- and 7D-infected dNPCs. (A) rOka-infected dNPCs. Normal VZV particles were observed in the nucleus (a1, capsids with genome, green arrow; capsids without genome, blue arrow) and in the cytoplasm and on the cell surface (a2, intact virion, green arrow; hollow particle, blue arrow), respectively. (B) 7D-infected dNPCs. 7D nucleocapsids were observed in the nucleus (b1 and b2, capsids with genome, green arrow; capsids without genome, blue arrow). Envelope-defective particles (red arrows) were captured in the cytoplasm (b3-b5). “Nu” and “Cy” indicate the nucleus and cytoplasm, respectively.

FIG 6

Viral particles in rOka- and 7D-infected dSY5Y cells. (A) rOka-infected dSY5Y cells. Normal VZV particles were observed in the nucleus (a1, capsids with genome, green arrow; capsids without genome, blue arrow), and in the cytoplasm (a2, intact virion, green arrow; hollow particle, blue arrow), respectively. (B) 7D-infected dSY5Y cells. 7D nucleocapsids were observed in the nucleus (b1 and b2, capsids with genome, green arrow; capsids without genome, blue arrow). Envelope-defective particles (red arrows) were captured in the cytoplasm (b3 to b5). “Nu” and “Cy” indicate the nucleus and the cytoplasm, respectively.

FIG 7

Viral particles in rOka- and 7D-infected ARPE-19 cells. (A) rOka-infected ARPE-19 cells. Normal VZV particles were observed in the nucleus (a1 and a2, capsids with genome, green arrow; capsids without genome, blue arrow) and in the cytoplasm and on the cell surface (a3 and a4, intact virion, green arrow; hollow particle, blue arrow), respectively. (B) 7D-infected ARPE-19 cells. 7D nucleocapsids were observed in the nucleus (b1 and b2, capsids with genome, green arrow; capsids without genome, blue arrow). Envelope-defective particles (red arrows) were captured in the cytoplasm and on the cell surface (b3 and b4). “Nu” and “Cy” indicate the nucleus and the cytoplasm, respectively.

FIG 8

Viral particles in rOka- and 7D-infected NPCs. (A) rOka-infected NPCs. Normal VZV particles were observed in the nucleus (a1 and a2, capsids with genome, green arrow; capsids without genome, blue arrow) and in the cytoplasm and on the cell surface (a3 to a5, intact virion, green arrow; hollow particle, blue arrow), respectively. (B) 7D-infected NPCs. 7D nucleocapsids were observed in the nucleus (b1 and b2, capsids with genome, green arrow). Envelope-defective particles (red arrows) were captured in the cytoplasm (b3 to b6). “Nu” and “Cy” indicate the nucleus and the cytoplasm, respectively.

FIG 9

Particles in cytoplasm of rOka- and 7D-infected cells. ARPE-19 cells (A), NPCs (B), dNPCs (C), and dSY5Y cells (D) were infected with rOka and 7D. Cytoplasmic viral particles were counted from five random infected cells from three independent experiments. Hollow particles, particles with a viral envelope but no capsid core (blue column); envelope-defective particles, particles with a capsid core but without complete envelope (red column); intact virions, particles with complete envelope, tegument layer, and capsid core (green column). No intact virions were observed in 7D-infected dNPCs and dSY5Y cells.