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. 2020 Apr 10;12(4):429. doi: 10.3390/v12040429

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Alphaherpesvirus gB localizes to EVs-enriched fractions isolated from virus-infected MJS cells and BoHV-1-infected MDBK. OptiPrep gradient-purification of EVs-enriched and virion-enriched fractions from HSV-1-infected MJS (A), BoHV-1-infected MJS (B), PRV-infected MJS (C), and BoHV-1-infected MDBK (D) supernatants. MJS cells were infected with HSV-1 at a multiplicity of infection (moi) of 0.1, with BoHV-1/PRV at an moi of 0.5; MDBK cells were infected at an moi of 0.1. After 50 h post-infection, 0.5 mL fractions were collected from 6%–18% iodixanol (Optiprep) gradient by ultracentrifugation. The 40 µL samples were analyzed in denaturing (for viral glycoprotein C-gC, glycoprotein B-gB, glycoprotein D-gD, for HSV-1, glycoprotein E-gE, for BoHV-1 and PRV or EVs marker Alix) or non-denaturing (for EVs markers CD63 and CD9) SDS-PAGE, and immunoblotted with specific mAbs or polyclonal anti-PRV gB serum. Uninfected (Ø) or virus-infected cell lysates (cl) were analyzed as controls. Bottom panels represent virus titres in each fraction measured by plaque assay. (E) Proteins from the indicated fractions (40 μL) were separated by SDS-PAGE, and immunoblotted with antibodies specific to the ICP5/VCP5 homolog of HSV-1 and BoHV-1, virulence factor γ34.5 from HSV-1, immunomodulatory UL49.5 protein of BoHV-1 and envelope glycoproteins gD, and gM, as indicated. HLA-DRβ was detected in the fractions as an EVs marker. Cell lysates (cl) and virus-infected cell lysates (30 μg) were analyzed as controls. (F) Comparison of HLA-DRβ levels between EVs from virus-infected and uninfected MJS. DRβ and flotillin-2 (flot-2, EVs marker) from F11 fractions were immunoblotted with specific mAb. Numbers indicate the optical density of DRβ bands from virus-infected MJS EVs, normalized to flot-2 from the same samples, relative to DRβ in EVs from uninfected MJS (set as 1).

Alphaherpesvirus gB localizes to EVs-enriched fractions isolated from virus-infected MJS cells and BoHV-1-infected MDBK. OptiPrep gradient-purification of EVs-enriched and virion-enriched fractions from HSV-1-infected MJS (A), BoHV-1-infected MJS (B), PRV-infected MJS (C), and BoHV-1-infected MDBK (D) supernatants. MJS cells were infected with HSV-1 at a multiplicity of infection (moi) of 0.1, with BoHV-1/PRV at an moi of 0.5; MDBK cells were infected at an moi of 0.1. After 50 h post-infection, 0.5 mL fractions were collected from 6%–18% iodixanol (Optiprep) gradient by ultracentrifugation. The 40 µL samples were analyzed in denaturing (for viral glycoprotein C-gC, glycoprotein B-gB, glycoprotein D-gD, for HSV-1, glycoprotein E-gE, for BoHV-1 and PRV or EVs marker Alix) or non-denaturing (for EVs markers CD63 and CD9) SDS-PAGE, and immunoblotted with specific mAbs or polyclonal anti-PRV gB serum. Uninfected (Ø) or virus-infected cell lysates (cl) were analyzed as controls. Bottom panels represent virus titres in each fraction measured by plaque assay. (E) Proteins from the indicated fractions (40 μL) were separated by SDS-PAGE, and immunoblotted with antibodies specific to the ICP5/VCP5 homolog of HSV-1 and BoHV-1, virulence factor γ34.5 from HSV-1, immunomodulatory UL49.5 protein of BoHV-1 and envelope glycoproteins gD, and gM, as indicated. HLA-DRβ was detected in the fractions as an EVs marker. Cell lysates (cl) and virus-infected cell lysates (30 μg) were analyzed as controls. (F) Comparison of HLA-DRβ levels between EVs from virus-infected and uninfected MJS. DRβ and flotillin-2 (flot-2, EVs marker) from F11 fractions were immunoblotted with specific mAb. Numbers indicate the optical density of DRβ bands from virus-infected MJS EVs, normalized to flot-2 from the same samples, relative to DRβ in EVs from uninfected MJS (set as 1).