Varicella-zoster virus ORF63 inhibits apoptosis of primary human neurons

Abstract

Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOka deltaORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.

FIG. 1.

Percentage of neurons displaying viral antigen expression in PAA-treated and untreated VZV-infected neurons by immunofluorescence analysis. At D1 and D2 p.i., neurons were incubated with rabbit polyclonal antibodies to IE gene products (IE62 and ORF4) and E gene products (ORF29) and with a hyperimmune serum that predominantly detects late-phase genes (glycoproteins). Secondary antibodies were FITC conjugated. Each column represents the mean ± SEM of viral antigen expression in three independent experiments derived from three different neuronal cultures.

FIG. 2.

Immunofluorescence analysis of viral antigen expression in rOkaΔORF63-infected neurons. At D1, D2, and D4 p.i., neurons were incubated with rabbit polyclonal antibody to an IE gene product (IE62) and a hyperimmune serum that predominantly detects late-phase gene products (glycoproteins). Secondary antibodies were FITC conjugated. Each column represents the mean ± SEM of viral antigen expression in three independent experiments derived from three different neuronal cultures.

FIG. 3.

Immunofluorescence analysis of apoptosis by annexin V staining and TUNEL in rOkaΔORF63-infected neurons. At D2 p.i., neurons infected with either rOka (A, B and E, G and I) or rOkaΔORF63 (C, D, F, H and J) were stained for annexin V (red fluorescence) and nuclear counterstain DAPI (blue fluorescence) (A to D) or subjected to TUNEL staining (green fluorescence) and propidium iodide staining (red fluorescence) (E to J). Positive control rOka-infected (A) and rOkaΔORF63-infected (C) neurons are shown. No specific annexin V staining was detected in rOka-infected neuronal cultures (B), while rOkaΔORF63-infected neuronal cultures (D) were positive. rOka-infected (E, G, I) or rOkaΔORF63-infected (F, H, J) neurons were harvested on day 2 p.i. No TUNEL-specific staining was detected in rOka-infected neuronal cultures (G). Positive TUNEL staining is shown in rOkaΔORF63-infected neuronal cultures (H). Positive controls (E and F) and negative controls (I and J) are shown. Bars, 10 μm.

FIG. 4.

Quantitation of apoptotic cells by use of annexin V and TUNEL in rOkaΔORF63-infected neurons. Percentage of neurons in mock-infected (Mock), rOka-, rOkaΔORF63-, and rOkaΔORF70-infected neuronal cultures stained for annexin V (A) and TUNEL apoptotic nuclei (B) over the 4-day time course.

FIG. 5.

Transmission electron micrographs of rOka-infected and rOkaΔORF63-infected human DRG neurons. (A) rOka-infected neuron displaying normal morphology. (B) rOkaΔORF63-infected neuron showing features of apoptosis. (C) Magnified unenveloped virion in rOka-infected neuron. (D) Magnified axonal cross section in rOkaΔORF63-infected neuron. (E) Magnified enveloped virion in cytoplasm of rOkaΔORF63-infected neuron.