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. 2018 Feb 26;92(6):e01842-17.
doi: 10.1128/JVI.01842-17. Print 2018 Mar 15.

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes

Farhana Musarrat  1 , Nithya Jambunathan  1 , Paul J F Rider  1 , V N Chouljenko  1 , K G Kousoulas  2
Affiliations

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes

Farhana Musarrat et al. J Virol. .

Abstract

Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.IMPORTANCE HSV-1 glycoprotein B (gB) functions in the fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), the release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.

Keywords: Akt; HSV-1; calcium signaling; fusion; glycoprotein B (gB); glycoprotein K (gK).

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Figures

FIG 1
FIG 1
Effect of an Akt inhibitor on HSV-1 entry. (A) Vero cells were treated with a series of dilutions of the Akt inhibitor miltefosine (labeled Akt inhibitor) for 15 min and then adsorbed with McKrae and McKrae gKΔ31-68 (100 PFU) for 1 h at 4°C. After incubation at 37°C for 48 h, the plaques (the number of PFU) were counted using crystal violet staining. *, P < 0.05 between McKrae and gKΔ31-68 viruses; ***, P < 0.001 versus no-drug-treated control; ns, no significance versus no-drug-treated control. Statistical comparison was conducted by GraphPad Prism software using ANOVA with a post hoc t test with the Bonferroni adjustment. Bars represent 95% confidence intervals about the means. (B) SK-N-SH cells were treated with miltefosine for 15 min and infected with McKrae or McKrae gKΔ31-68 (MOI = 10) for 1 h at 37°C, and the proximity ligation assay (PLA) was performed. Confocal microscopy was used to detect bright red spots, which indicate an interaction between two proteins after drug treatment, at a magnification of ×63 with oil immersion. The interaction between UL37 (capsid protein) and dynein (cellular protein) was used as a measure of entry of the virus. The interaction between gD and nectin-1 was used as a positive PLA control in this experiment. DAPI was used to stain the nuclei of the cells.
FIG 2
FIG 2
Interaction between gB and Akt-1(S473). (A) Proximity ligation assay showing the interaction between gB and Akt-1(S473) in McKrae- and McKrae gKΔ31-68-infected SK-N-SH cells at 1 h postinfection at an MOI of 10. The gD–nectin-1 interaction was used as a positive control, and the gD–Akt-1(S473) interaction was used as a negative control. Confocal microscopy was used to detect the bright red spots that suggest the interaction between two proteins. DAPI was used to stain the nuclei of the cells. Magnifications, ×63 with oil immersion. (B) Two-way immunoprecipitation (IP) showing the gB–Akt-1(S473) interaction in McKrae and McKrae gKΔ31-68 virus-infected (MOI = 10) SK-N-SH cell lysates.
FIG 3
FIG 3
HSV-1 triggers intracellular calcium release. Vero cells (A) and SK-N-SH cells (B) were grown in a 96-well black clear-bottom plate and treated with Fura-2AM (25 μM) for 30 min at 37°C. After washing, the cells were adsorbed with purified McKrae and McKrae gKΔ31-68 (both at an MOI of 10) at 4°C for 1 h and then shifted to 37°C to measure the absorbance every minute over an hour. The ratio of bound to unbound dye (340/380 nm) was measured, the intracellular calcium concentration was calculated, and the average values are presented. Ionomycin was used as a positive control, and 1× PBS was used as a negative control. ***, P < 0.001 versus the PBS control; ns, no significance versus the PBS control. Statistical comparison was conducted by GraphPad Prism software using ANOVA with a post hoc t test with the Bonferroni adjustment. Bars represent the 95% confidence intervals about the means.
FIG 4
FIG 4
Phosphatidylserine exposure on cell surface. (A) Vero cells were adsorbed with either McKrae or McKrae gKΔ31-68 at an MOI of 10 for 1 h at 4°C, and then the 8-well chamber slides were shifted to 37°C for 5 min. Cells were live stained with FITC-conjugated annexin V to detect phosphatidylserine on the surface. Fluorescent (FITC), phase-contrast, and merged images of the infected and uninfected control Vero cells are presented. A fluorescence microscope was used to take the images. Magnifications, ×40. (B) The number of cells labeled by FITC-conjugated annexin V per area of 100 mm2 was counted. Values were normalized to those for the uninfected control wells. Statistical comparison was conducted by GraphPad Prism software using Student's t test. Bars represent the 95% confidence intervals about the means. A comparison was made between the viruses.
FIG 5
FIG 5
Akt-1(S473) exposure on the cell surface. (A) Vero cells were infected with either McKrae VP26-EGFP or McKrae gKΔ31-68 VP26-EGFP at an MOI of 10 for 30 min at 37°C and were then reacted with rabbit anti-Akt-1(S473) antibody (1:100) under live conditions for 30 min at 37°C. After acid washing, the cells were formalin fixed and stained with anti-rabbit immunoglobulin antibody conjugated with Alexa Fluor 594 (red) and plasma membrane stain (purple). The slides were mounted with mounting medium with DAPI (cyan). (B) (Top) Vero cells were either reacted with mouse antidynein antibody and formalin fixed (left) or fixed and permeabilized with methanol and reacted with mouse antidynein antibody for 30 min at 37°C (right). (Bottom) Cells were washed with PBS and stained with anti-mouse immunoglobulin antibody conjugated with Alexa Fluor 594 (red) and were mounted with mounting medium with DAPI. (C) Vero cells were infected with either McKrae VP26-EGFP or McKrae gKΔ31-68 VP26-EGFP for an hour at 37°C and then fixed and permeabilized with methanol. After washing, the cells were reacted with rabbit anti-Akt-1(S473) antibody for 30 min at 37°C and stained with anti-rabbit immunoglobulin antibody conjugated with Alexa Fluor 594 (red). The slides were mounted with mounting medium with DAPI. Confocal microscopy was used to take the images. Magnifications, ×63 with oil immersion.
FIG 6
FIG 6
Endocytosis assay. (A) SK-N-SH cells were treated with no drug, 30 μM pitstop-2, or 80 μM dynasore hydrate or with 30 μM pitstop-2 plus 80 μM dynasore hydrate in combination for 30 min at 37°C. Cells were infected with either McKrae or McKrae gKΔ31-68 for 1 h at an MOI of 10 at 37°C and then prepared for PLA. The UL37-dynein interaction was used as a measure of entry of the viral capsid into the cell, and the gD–nectin-1 interaction was used as a PLA control. Confocal microscopy was used to detect the bright red spots that suggest the interaction between two proteins. DAPI was used to stain the nuclei of the cells. Magnifications 63× with oil immersion. (B) Vero cells were treated with combined doses of pitstop-2 at 10, 20, 30, or 50 μM (P10, P20, P30 and P50, respectively) and dynasore hydrate at 80 μM (D80) for 30 min at 37°C and then infected with 100 PFU the McKrae or McKrae gKΔ31-68 virus for 1 h at 37°C (without washing the drug off). After infection, the wells were washed with PBS and methylcellulose was added. The plaques (the numbers of PFU) were counted using crystal violet staining after 48 h. *, P < 0.05 versus the no-drug-treated control (ND); ***, P < 0.001 versus the no-drug-treated control. Statistical comparison was conducted by GraphPad Prism software using ANOVA with a post hoc t test with the Bonferroni adjustment. Bars represent the 95% confidence intervals about the means.
FIG 7
FIG 7
Model of HSV-1 entry into host cells. The schematic shows the molecules involved in HSV-1 entry via either endocytosis or fusion between the viral envelope and the cellular plasma membrane as well as the intracellular signaling that may be involved with these two entry pathways.
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References

    1. Roizman B, Knipe DM. 2001. Herpes simplex viruses and their replication, p 2399–2459. In Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE (ed), Fields virology, 3rd ed, vol 2 Lippincott-Williams & Wilkins, Philadelphia, PA.
    1. Aggarwal A, Miranda-Saksena M, Boadle RA, Kelly BJ, Diefenbach RJ, Alam W, Cunningham AL. 2012. Ultrastructural visualization of individual tegument protein dissociation during entry of herpes simplex virus 1 into human and rat dorsal root ganglion neurons. J Virol 86:6123–6137. doi:10.1128/JVI.07016-11. - DOI - PMC - PubMed
    1. Nicola AV. 2016. Herpesvirus entry into host cells mediated by endosomal low pH. Traffic 17:965–975. doi:10.1111/tra.12408. - DOI - PMC - PubMed
    1. Eisenberg RJ, Atanasiu D, Cairns TM, Gallagher JR, Krummenacher C, Cohen GH. 2012. Herpes virus fusion and entry: a story with many characters. Viruses 4:800–832. doi:10.3390/v4050800. - DOI - PMC - PubMed
    1. Lycke E, Hamark B, Johansson M, Krotochwil A, Lycke J, Svennerholm B. 1988. Herpes simplex virus infection of the human sensory neuron. An electron microscopy study. Arch Virol 101:87–104. - PubMed

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