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. 2006 Nov;80(21):10325-34.
doi: 10.1128/JVI.00939-06.

Postentry events are responsible for restriction of productive varicella-zoster virus infection in Chinese hamster ovary cells

Renée L Finnen  1 , Kara R Mizokami, Bruce W Banfield, Guang-Yun Cai, Scott A Simpson, Lewis I Pizer, Myron J Levin
Affiliations

Postentry events are responsible for restriction of productive varicella-zoster virus infection in Chinese hamster ovary cells

Renée L Finnen et al. J Virol. 2006 Nov.

Abstract

Productive infection of varicella-zoster virus (VZV) in vitro is restricted almost exclusively to cells derived from humans and other primates. We demonstrate that the restriction of productive VZV infection in CHO-K1 cells occurs downstream of virus entry. Entry of VZV into CHO-K1 cells was characterized by utilizing an ICP4/beta-galactosidase reporter gene that has been used previously to study herpes simplex virus type 1 entry. Entry of VZV into CHO-K1 cells involved cell surface interactions with heparan sulfate glycosaminoglycans and a cation-independent mannose-6-phosphate receptor. Lysosomotropic agents inhibited the entry of VZV into CHO-K1 cells, consistent with a low-pH-dependent endocytic mechanism of entry. Infection of CHO-K1 cells by VZV resulted in the production of both immediate early and late gene products, indicating that a block to progeny virus production occurs after the initiation of virus gene expression.

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Figures

FIG. 1.
FIG. 1.
Blue foci form on cultured rodent cells following inoculation with lacZ-expressing VZV. (A to D) Monolayers of fully permissive MeWo cells (A) and the rodent cell lines NIH 3T3 (B), CHO-K1 (C), and R3A (D) were inoculated with CF ROka-lacZ and stained at 24 hpi with Bluo-Gal. (E to G) Monolayers of NIH 3T3 cells grown in six-well dishes were inoculated with CF ROka-lacZ, stained at 72 hpi with Bluo-Gal (E), and subsequently stained with goat polyclonal antisera against VZV IE62 and an Alexa fluor 555-conjugated rabbit anti-goat secondary antibody (F). Merged signals are shown in panel G.
FIG. 2.
FIG. 2.
Inoculation of CHO-IEβ8 cells with CF VZV results in increased β-Gal production. CHO-IEβ8 cells were inoculated with CF or CA POka at an MOI of 0.05. Mock-treated cells were inoculated with a lysate prepared from uninfected cells. Cell extracts were prepared at the indicated times postinfection and assayed for β-Gal production. The experiment was performed in triplicate.
FIG. 3.
FIG. 3.
Removal of virus from CF inoculum results in decreased β-Gal production. Virus was removed from the CF POka inoculum by centrifugation, and the supernatant was used to inoculate CHO-IEβ8 cells alongside the noncentrifuged CF inoculum. Cell extracts were prepared at 0 and 18 hpi and assayed for β-Gal production. The experiment was performed in triplicate. The dashed line indicates the increase in β-Gal production expected from cell growth alone.
FIG. 4.
FIG. 4.
Inactivation of CF VZV by brief treatment with acidic pH results in decreased β-Gal production. (A) CF POka was treated with 800 mM sodium citrate, pH 2.2 or 7.2, neutralized by dilution in medium, and then assayed for plaque production on MeWo cells. The average number of plaques for four separate wells was determined and scored relative to that for untreated, similarly diluted CF POka. (B) CF POka was allowed to bind to chilled CHO-IEβ8 cells at 4°C, treated briefly with warm medium at various pHs, and then incubated in regular medium. Cell extracts were prepared at 18 hpi and assayed for β-Gal production. The β-Gal production values were scored relative to those for control samples treated with unadjusted medium (set to 100%). The experiment was performed in triplicate. (C) CF POka was allowed to bind to chilled CHO-IEβ8 cells at 4°C and treated briefly with warm sodium citrate, pH 3.68, following a 0-, 0.5-, or 2-h incubation in regular medium at 37°C. Cell extracts were prepared at 18 hpi and assayed for β-Gal production. The β-Gal production values were scored relative to those for samples that were subjected to pH inactivation at 2 hpi (set to 100%). The experiment was performed in quadruplicate. Dashed lines in panels B and C indicate the increase in β-Gal production expected from cell growth alone.
FIG. 5.
FIG. 5.
Agents known to block VZV-cell surface interactions affect entry of VZV into CHO-K1 cells. CHO-IEβ8 cells were pretreated for 30 min with the indicated concentrations of heparin (A), M6P (B), and G1P (B) and then inoculated with CF POka. Infections were carried out in the continual presence of the agent under study. G1P was included as a specificity control for M6P blocking. Cell extracts were prepared at 18 hpi and assayed for β-Gal production. The β-Gal production values were scored relative to those for untreated control samples (set to 100%). Experiments for both panels were performed in triplicate. Dashed lines indicate the increase in β-Gal production expected from cell growth alone.
FIG. 6.
FIG. 6.
Inhibitors of endosome acidification affect the entry of VZV into CHO-IEβ8 cells. CHO-IEβ8 cells were pretreated with an endosome acidification inhibitor at the indicated concentrations. CF POka was allowed to bind to chilled CHO-IEβ8 cells at 4°C, and warm inhibitor-containing medium was added following a 0- or 1-h incubation in regular medium at 37°C. Inhibitor-containing medium was removed at 2.5 hpi or 3.5 hpi (panel B, light gray bars), noninternalized virus was inactivated, and incubation was continued in regular medium. Cell extracts were prepared at 21 hpi and assayed for β-Gal production. The β-Gal production values were scored relative to those for untreated, similarly processed control samples (set to 100%). The results shown in both panels are for two independent experiments, each of which was performed in triplicate. Dashed lines indicate the increase in β-Gal production expected from cell growth alone.
FIG. 7.
FIG. 7.
Presence of PAA results in decreased β-Gal production. CHO-IEβ8 cells were pretreated with 300 μg/ml PAA and then inoculated with CF POka. Infections were carried out in the constant presence of PAA. Cell extracts were prepared at 18 hpi and assayed for β-Gal production. The β-Gal production values in the presence of PAA were scored relative to those for untreated, similarly processed controls (set to 100%). The experiment was performed in triplicate. The dashed line indicates the increase in β-Gal production expected from cell growth alone.
FIG. 8.
FIG. 8.
VZV late protein gE is produced in infected CHO-K1 cells. Indirect immunofluorescence microscopy images of MeWo and CHO-K1 cells following infection with CF POka are shown. Cells were stained with mouse anti-IE62 monoclonal antibodies, goat anti-gE polyclonal antiserum, and conjugated secondary antisera (Alexa fluor 488-conjugated donkey anti-mouse for IE62 and Alexa fluor 555-conjugated rabbit anti-goat for gE); nuclei were stained with Hoechst reagent. Representative fields of infected MeWo cells at 6 hpi and 24 hpi are shown in panels A to D and E to H, respectively; representative fields of infected CHO-K1 cells at 6 hpi or 24 hpi are shown in panels I to L and M to P, respectively. Panels A, E, I, and M show IE62 (ORF62) staining, panels B, F, J, and N show gE (ORF68) staining, and panels C, G, K, and O show Hoechst staining. Merged signals are shown in panels D, H, L, and P.
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References

    1. Annunziato, P., P. LaRussa, P. Lee, S. Steinberg, O. Lungu, A. A. Gershon, and S. Silverstein. 1998. Evidence of latent varicella-zoster virus in rat dorsal root ganglia. J. Infect. Dis. 178(Suppl. 1):S48-S51. - PubMed
    1. Banfield, B. W., Y. Leduc, L. Esford, K. Schubert, and F. Tufaro. 1995. Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway. J. Virol. 69:3290-3298. - PMC - PubMed
    1. Banfield, B. W., Y. Leduc, L. Esford, R. J. Visalli, C. R. Brandt, and F. Tufaro. 1995. Evidence for an interaction of herpes simplex virus with chondroitin sulfate proteoglycans during infection. Virology 208:531-539. - PubMed
    1. Bourdon-Wouters, C., M. P. Merville-Louis, C. Sadzot-Delvaux, P. Marc, J. Piette, P. Delree, G. Moonen, and B. Rentier. 1990. Acute and persistent varicella-zoster virus infection of human and murine neuroblastoma cell lines. J. Neurosci. Res. 26:90-97. - PubMed
    1. Browne, H., B. Bruun, and T. Minson. 2001. Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL. J. Gen. Virol. 82:1419-1422. - PubMed

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