Fig 1: A small-molecule OST inhibitor impaired the propagation of the rLCMV/LASV GPC virus. (A) STT3A–, STT3B–, MAGT1– TUSC3–, and WT (HEK293T) cells were infected with the rLCMV/LASV GPC virus at an MOI of 0.01. NGI-1 at the indicated concentrations was added to cells after virus entry. Dimethyl sulfoxide (DMSO) was added as a control. Cells and supernatants were harvested at 36 hpi. The glycosylation patterns of viral glycoprotein were determined by Western blotting with GP2-specific serum. GAPDH was also detected as a loading control for each lane. (B) Supernatant viral titers and viral RNA copy numbers were determined by the immunological plaque assay and qRT-PCR, respectively. Viral titers less than 10 PFU/ml were considered undetected and are marked with a red number sign (“#”). (C) A549, HeLa, Huh7, and HEK293 cells were infected with the rLCMV/LASV GPC virus at an MOI of 0.01. NGI-1 at the indicated concentrations were added to cells after virus entry. DMSO was added as a control. Cells and supernatants were harvested at 36 hpi. The glycosylation patterns of viral glycoprotein were determined by Western blotting with GP2-specific serum. GAPDH was also detected as a loading control for each lane. (D) Supernatant viral titers were determined by the immunological plaque assay and qRT-PCR, respectively. Viral titers of <10 PFU/ml were considered undetected and are marked with a red number sign (“#”).
Fig 2: Knockout of STT3A or STT3B led to the formation of viral particles with reduced infectivity. (A) Single-step infection assay was performed to assess the productive entry and early infection of the rLCMV/LASV GPC virus in STT3A– and STT3B– cells. The cells were infected with virus at an MOI of 0.01, incubated for 30 min at 4°C to synchronize the entry process, and then shifted to 37°C to allow penetration. Cells were harvested at 8 hpi, and the viral NP level was detected by Western blotting with anti-NP serum. (B) Intracellular and supernatant genome virus levels were measured at 48 hpi to assess virus budding ability by qRT-PCR. The ratio of the supernatant viral genome level versus the intracellular viral genome level was calculated as an indicator of virus budding ability (presented above the column). (C) The relative PFU in the supernatants of different cells at 48 hpi are shown. (D) The ratios of viral PFU versus viral genome RNA copy numbers were calculated as an indicator of viral infectivity. All data displayed here represent the means ± the standard deviations (SD) of three independent experiments, and each independent experiment had two replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Viral foci were observed by fluorescence microscopy at different time points in cells infected with the rLCMV (NP-P2A-GFP)/LASV GPC virus. The foci in STT3A– and STT3B– cells were notably smaller than those in WT cells. All of the micrographs displayed here were collected at the same scale (the length of the red bar represents 100 µm) and are representatives of several images.
Fig 3: Knockout of STT3B caused hypoglycosylation of LASV GP and reduced its expression level. (A) Expression and glycosylation profiles of LASV GP in STT3A–, STT3B– cells and WT cells. Cells infected with the rLCMV/LASV GPC virus at an MOI of 0.01 were harvested at the indicated time points and subjected to Western blot analysis with GP2-specific serum. Viral NP was also detected by a specific antiserum, serving as an internal control to reflect the replication level of the virus. The cleavage rates of tagged and untagged GP2 are shown above as determined by densitometric quantification. (B) The reduced molecular weight of viral GP in STT3B– cells was due to a deficiency in glycans. Viral proteins from different cell lines were subjected to cleavage by PNGase F and Western blot analysis with GP2-specific serum. Viral NP was also detected. (C) Quantitation of viral glycoprotein incorporated into viral particles. Viral particles (supernatant) derived from different cell lines at 48 hpi were collected by ultracentrifugation and screened for viral proteins (GP1, GP2, and NP) by Western blotting. (D) The levels of incorporated proteins were determined by the band intensities, and the ratios of viral GP2 versus viral NP were calculated as an indicator of the level of viral glycoproteins incorporated into a single viral particle. SDs are not shown because viral particles were derived from one individual preparation. In panels A to C, GAPDH was also detected as a loading control for each lane. (E) The cell binding ability of RNA-normalized viruses derived from different cell lines to normal BHK-21 cells was measured by qRT-PCR and normalized to the GAPDH mRNA level. The data displayed here represent the means ± the SD of three independent experiments, and each independent experiment had two replicates.
Fig 4: The preferential requirement for STT3B-dependent N-glycosylation was conserved among arenavirus GPs. pCAGGs plasmids encoding Strep-tagged glycoproteins of four OW arenaviruses (A) and two NW arenaviruses (B) or VSV-G, influenza A virus HA, and HIV-1 GP160 (C) were transfected into STT3A–, STT3B–, MAGT1– TUSC3–, and WT cells. Cells were harvested at 24 h posttransfection and subjected to Western blot analysis with antibodies specific to the Strep epitope. Hypoglycosylation patterns of virus glycoprotein are indicated by red triangles. GAPDH was also detected as a loading control for each lane. (D) The supernatant viral titers and viral RNA copy numbers of LCMV and rLCMV/MACV GPC viruses in STT3A–, STT3B–, MAGT1– TUSC3–, and WT cells were determined by the immunological plaque assay and qRT-PCR, respectively. (E) The supernatant virus titers and viral RNA copy numbers of the LCMV and rLCMV/MACV GPC viruses in HEK293T cells treated with the indicated concentrations of NGI-1. DMSO was added as a control. Viral titers of <10 PFU/ml were considered undetected and are marked with a red number sign (“#”). (F) Viral foci were observed by fluorescence microscopy at 36 hpi in cells infected with the rLCMV (NP-P2A-GFP) and rLCMV (NP-P2A-GFP)/MACV GPC viruses. The viral foci in OST-knockout cells, especially in STT3B– and MAGT1– TUSC3– cells, were notably smaller than those in WT cells. All the micrographs displayed here were collected at the same scale (the length of the red bar represents 100 µm) and are representatives of several images.
Fig 5: Confirmation of the interactions between LASV GP and subunits of the OST complex by coimmunoprecipitation and Western blot analyses. HEK293T cells were transfected with a pCAGGs plasmid encoding Twin-Strep-tagged LASV GP or GFP, and at 48 h posttransfection the cells were harvested and lysed at 4°C. LASV GP and GFP were pulled down from the cell lysates by magnetic Sepharose beads coated with Strep-Tactin XT as bait (Input). Purified bead fractions were screened for endogenous STT3A (A) and STT3B (B) by SDS-PAGE and Western blot analysis using antibodies specific to human STT3A and STT3B, respectively. (C to H) A pCAGGs plasmid encoding V5-tagged RPN1, RPN2, OSTC, or DDOST; 6×His-tagged MAGT1; or Flag-tagged TUSC3 was cotransfected into HEK293T cells with a plasmid expressing LASV GP or GFP. Purified bead fractions were screened for RPN1 (C), RPN2 (D), OSTC (E), DDOST (F), MAGT1 (G), and TUSC3 (H) by SDS-PAGE and Western blot analysis with antibodies specific to the V5 (RPN1, RPN2, OSTC, and DDOST), 6×His (MAGT1) or Flag (TUSC3) epitopes. LASV GP and GFP were detected by an anti-Strep antibody (A to H, left). GAPDH was also detected as a control of cell lysate input and bead fraction output (A to H, bottom). Bands indicating specific interactions between LASV GP and the OST subunits are marked with a red star. Note that some of the OST subunits were found to migrated into diffused bands and formed high-molecular-weight aggregates in the SDS-PAGE gels. LASV GP but not the control GFP was able to pull down all of the subunits identified in our MS data set.
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