Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells

Figures

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  Figure 1. Schematic of the recombinant equine arteritis virus (EAV) construction (A). Introduction of the ORF4 Gp4 start codon mutation (1, green star) and the AscI nucleotide sequence (2, arrow). Separation of the overlapping ORF3 and ORF4 by introducing the beginning of the Gp4 sequence (3). Addition of the HA-tag sequence to the end of ORF3 (4). Expression of Gp3-HA and N protein in BHK-21 cells (B). Cells were transfected with in vitro-transcribed RNA produced from pEAV211, pEAV211s3/4, and pEAV211Gp3-HA. After the appearance of the cytopathic effect, the cells were lysed and subjected to SDS-PAGE and western blotting with anti-HA and anti-N antibodies. The cells were then transfected with in vitro-transcribed RNA: pEAV211 (WT), lane 1; pEAV211s3/4, lane 2; pEAV211Gp3-HA, lane 3; and untransfected cells, lane 4. The apparent molecular masses in kDa are shown on the right side. Expression of Gp3-HA and N in infected BHK-21 cells (C). Cells were infected with culture supernatants from BHK-1 cells previously transfected with pEAV211 (WT), pEAV211s3/4, and pEAV211Gp3-HA. They were then subjected to immunofluorescence 24 h post-infection with anti-N and anti-HA antibodies. WT—cells infected…

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  Figure 2. Growth kinetics of the EAV recombinant viruses (A). BHK-21 cells were infected with an MOI of 0.1 of P1 stocks of EAV (wt) and recombinant EAVs3/4 and EAVGp3-HA viruses. Supernatants were collected at 6, 12, 24, 48 and 72 h post-infection and subjected to plaque assay. PFU/mL—plaque forming unit per mL. Expression of Gp3-HA is stable upon infection (B). EAVGp3-HA virus was passaged 19 times on fresh BHK-21 cells. Cells were infected with culture supernatants containing recombinant viruses from passages P7, P10, P15, and P19, and subjected to immunofluorescence assay with anti-HA-tag and anti-N antibodies after 18 h p.i. N is shown in red and Gp3-HA is shown in green. Cell nuclei were stained with DAPI. Chromatograms of viral DNA sequences obtained from EAVGp3-HA infected cells (C). Cells infected with P7 and P19 were subjected to RT-PCR. The fragment of the HA-tag nt sequence is shown. The consensus sequence was maintained, but the presence of additional nt peaks in P19 suggested the increase of quasispecies of the virus.

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  Figure 3. Characterization of Gp3-HA in infected cells (A). BHK-21 cells were infected with P1 EAVGp3-HA at an MOI of 1. Then, 18 h p.i. cells were washed, scraped from the dish, pelleted, and lysed in a non-reducing buffer (-DTT; left part of the blot) or in the deglycosylation buffer (right part of the blot). To analyze if the Gp3-HA is N-glycosylated in infected cells, a part of the samples was subjected to deglycosylation with endoglucosidase H (EH) or PNgase F (PF). The samples were then subjected to SDS-PAGE and western blotting with anti-HA antibodies. EAVGp3-HA - cells infected with EAVGp3-HA; mock - uninfected cells. The apparent molecular masses in kDa are shown on the right. Gp3-HA is present in the virions as a monomer and a trimer (B). To investigate whether the tagged Gp3-HA is incorporated into the virions, the BHK-21 cells were infected with P1 EAVGp3-HA stock at an MOI of 1. The supernatants (22 h p.i.) were collected, cleared with low-speed centrifugation, and concentrated on the filter devices. Major part (95%) of the purified virus sample was subjected to immunoprecipitation (IP) with anti-HA antibodies, and the remaining 5% of the sample was boiled with non-reducing or…

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  Figure 4. Gp3-HA partially co-localize with E protein (A). The BHK-21 cells were infected with EAVGp3-HA at an MOI of 1 or left uninfected. Then, the cells (18 h p.i.) were subjected to immunofluorescence with mouse anti-HA antibodies and rabbit anti-E antibodies and secondary Alexa Fluor 568-conjugated anti-mouse antibodies and Alexa Fluor 488-conjugated anti-rabbit antibodies, respectively. E is shown in green, Gp3-HA is shown in red. Gp3-HA shows little co-localization with N protein (B). Cells were infected as above; Cells (18 h p.i.) were then subjected to immunofluorescence with rabbit anti-HA antibodies and mouse anti-N antibodies and secondary Alexa Fluor 568-conjugated anti-mouse antibodies and Alexa Fluor 488-conjugated anti-rabbit antibodies, respectively. N is shown in red, Gp3-HA is shown in green. E shows little co-localization with N protein (C). Cells were infected as above; Cells (18 h p.i.) were then subjected to immunofluorescence with rabbit anti-E antibodies and mouse anti-N antibodies and secondary Alexa Fluor 568-conjugated anti-mouse antibodies and Alexa Fluor 488-conjugated anti-rabbit antibodies, respectively. N is shown in red, E is shown in green. The cell…

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  Figure 5. Co-localization of Gp3-HA with markers of the secretory pathway. The BHK-21 cells were infected with EAVGp3-HA at an MOI of 1 or left uninfected. Cells (18 h p.i.) were then subjected to immunofluorescence. Gp3-HA co-localized with PDI, marker for ER (A). Confocal analysis of the immunofluorescence samples stained with rabbit anti-HA antibodies and mouse anti-PDI antibodies and secondary Alexa Fluor 568-conjugated anti-mouse antibody and Alexa Fluor 488-conjugated anti-rabbit antibodies, respectively. Gp3-HA is shown in green, PDI is shown in red. Gp3-HA minimally co-localized with ERGIC (B). Confocal analysis of the immunofluorescence samples stained with rabbit anti-HA antibodies and mouse anti-ERGIC53 antibodies and secondary antibodies Alexa Fluor 568 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies, respectively. Gp3-HA is shown in green, ERGIC is shown in red. Gp3-HA co-localizes with membrin, marker for cis-Golgi (C). Confocal analysis of the immunofluorescence samples stained with rabbit anti-HA antibodies and mouse anti-membrin antibodies and secondary Alexa Fluor 568-conjugated anti-mouse antibody and Alexa Fluor 488-conjugated anti-rabbit antibodies, respectively.…

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  Figure 6. Co-localization of E with markers of the secretory pathway. The BHK-21 cells were infected with EAVGp3-HA at an MOI of 1 or left uninfected. The cells (18 h p.i.) were then subjected to immunofluorescence. E partially co-localizes with PDI, marker for ER (A). Confocal analysis of the immunofluorescence samples stained with rabbit anti-E antibodies and mouse anti-PDI antibodies and secondary antibodies Alexa Fluor 568 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies. E is shown in green, PDI is shown in red. E minimally co-localizes with ERGIC (B). Confocal analysis of the immunofluorescence samples stained with rabbit anti-E antibodies and mouse anti-ERGIC53 antibody and secondary antibodies Alexa Fluor 568 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies, respectively. E is shown in green, ERGIC is shown in red. E partially co-localizes with membrin, marker for cis-Golgi (C). Confocal analysis of the immunofluorescence samples stained with rabbit anti-E antibodies and mouse anti-membrin antibodies and secondary antibodies Alexa Fluor 568 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies. E is shown in green, membrin is shown in red. The cell nuclei were stained…