Journal: Cell Reports

Article Title: Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation

doi: 10.1016/j.celrep.2020.108235

Figure Lengend Snippet: Identification of pUL56 Degradation Targets (A) HaCaT cells were infected with HSV-1 WT and HSV-1 ΔUL56 at an MOI of 10 in biological duplicates and total infectious virus yields at the indicated time points were determined by plaque assay. Error bars represent standard error of the mean. (B) Plaque assays of HSV-1 WT and HSV-1 ΔUL56 in HaCaT, HFF hTERT, and Vero cells in biological duplicates. Plaques were visualized by immunostaining the cells for the viral glycoprotein gD. (C) Plaque diameters from (B) were measured and normalized to the average for HSV-1 WT. Error bars represent standard deviation; n = 35–67. (D) Schematic of the proteomics workflow. Cells were infected at an MOI of 10 or mock infected. Samples were harvested at the stated times and processed for quantitative proteomic analysis. Data shown in . (E) Scatterplot of all proteins quantified. Fold changes were calculated for each protein by comparing S:N values at 8 hpi for HSV-1-WT- and HSV-1-ΔUL56-infected samples. Benjamini-Hochberg-corrected significance B was used to estimate p values . (F) Temporal profiles of all proteins downregulated >2-fold by HSV-1 WT versus mock and additionally rescued >2-fold by HSV-1 ΔUL56.

Article Snippet: The following mammalian cell lines were used in this work: HaCaT cells: human keratinocyte cell line, spontaneously immortalized, male ( ); HFF hTERT cells: human foreskin fibroblast cell line, telomerase immortalized, male ( ); Vero cells: African green monkey kidney cell line, spontaneously immortalized, female (ATCC, CRL-1586); HEK293T cells: human embryonic kidney cell line, Adenovirus 5 and SV40 transformed, female (ATCC, CRL-3216); U2-OS cells: human osteosarcoma cell line, cancer cell line, female (ATCC, HTB-96).

Techniques: Infection, Plaque Assay, Immunostaining, Standard Deviation

Journal: Cell Reports

Article Title: Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation

doi: 10.1016/j.celrep.2020.108235

Figure Lengend Snippet: pUL56 Is Necessary and Sufficient for GOPC Degradation (A) HaCaT cells were infected at an MOI of 10 with the indicated viruses. After 2 h, media were replaced with 10 μM MG132 or carrier (DMSO) in DMEM for the remainder of the infection. Cell lysates were harvested 16 hpi and the indicated proteins were detected by immunoblot. (B) HFF hTERT cells were infected at an MOI of 1 and then treated with MG132 or carrier as described in (A). At 6 hpi, samples were fixed and stained for GOPC (green) and the infection control gD (red). The merge includes DAPI (blue). The scale bar represents 10 μm. Asterisks indicate HSV-1 infected (gD expressing) cells. (C) U2-OS cells were transfected with GFP-pUL56 or GFP-pUL56-AAXA expression plasmids. One day post-transfection, cells were fixed and stained for GOPC (red) and TGN46 (cyan). The merge includes DAPI (blue). The scale bar represents 10 μm. Asterisks indicate GFP-pUL56 and GFP-pUL56 AAXA expressing cells. (D) HaCaT cells were infected at an MOI of 10 with the indicated virus, cell lysates were harvested 16 hpi, and the indicated proteins were detected by immunoblot. (E) HEK293T cells were transfected with YFP-tagged NEDD4-WW domains, myc-tagged GOPC, and untagged pUL56 or pUL56-AAXA expression plasmids. Samples were subjected to IP using YFP affinity resin and co-precipitated proteins were detected by immunoblot. (F) HEK293T cells were transfected with HA-tagged ubiquitin (HA-Ub) and myc-GOPC together with empty vector or pUL56 or pUL56-AAXA expression plasmids. Samples were subjected to IP using myc affinity resin and probed for the presence of HA-Ub-conjugated GOPC by immunoblot.

Article Snippet: The following mammalian cell lines were used in this work: HaCaT cells: human keratinocyte cell line, spontaneously immortalized, male ( ); HFF hTERT cells: human foreskin fibroblast cell line, telomerase immortalized, male ( ); Vero cells: African green monkey kidney cell line, spontaneously immortalized, female (ATCC, CRL-1586); HEK293T cells: human embryonic kidney cell line, Adenovirus 5 and SV40 transformed, female (ATCC, CRL-3216); U2-OS cells: human osteosarcoma cell line, cancer cell line, female (ATCC, HTB-96).

Techniques: Infection, Western Blot, Staining, Expressing, Transfection, Plasmid Preparation

Journal: Cell Reports

Article Title: Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation

doi: 10.1016/j.celrep.2020.108235

Figure Lengend Snippet:

Article Snippet: The following mammalian cell lines were used in this work: HaCaT cells: human keratinocyte cell line, spontaneously immortalized, male ( ); HFF hTERT cells: human foreskin fibroblast cell line, telomerase immortalized, male ( ); Vero cells: African green monkey kidney cell line, spontaneously immortalized, female (ATCC, CRL-1586); HEK293T cells: human embryonic kidney cell line, Adenovirus 5 and SV40 transformed, female (ATCC, CRL-3216); U2-OS cells: human osteosarcoma cell line, cancer cell line, female (ATCC, HTB-96).

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Magnetic Beads, Staining, Microscopy, Bicinchoninic Acid Protein Assay, BIA-KA, Mass Spectrometry, CRISPR, Software

Journal: bioRxiv

Article Title: Herpes simplex virus-1 pUL56 degrades GOPC to alter the plasma membrane proteome

doi: 10.1101/729343

Figure Lengend Snippet: A . HaCaT cells were infected at MOI of 10 with the indicated virus. After 2 h, media was replaced with 10 μM MG132 or carrier (DMSO) in DMEM for the remainder of the infection. Cell lysates were harvested 16 hpi. B. HFF hTERT cells were infected at MOI of 1 then treated with MG132 or carrier as described in ( A ). At 6 hpi, samples were fixed and stained for GOPC (green) and the infection control gD (red). The merge includes DAPI (blue) and the scale bar represents 10 μm. C. HFF cells were transfected with pUL56-GFP. One day post-transfection cells were fixed and stained for GOPC (red). The merge includes DAPI (blue) and the scale bar represents 10 μm. D. Expression of pUL56 was induced with doxycycline in a clonal 293 FlpIn cell line. Cell lysates were harvested 1 day after induction. E. HaCaT cells were infected at MOI of 10 with the indicated virus and cell lysates were harvested 16 hpi.

Article Snippet: HaCaT , telomerase immortalized human foreskin fibroblasts HFF hTERT , Vero (ATCC), HEK293T (ATCC), and Flp-In™-293 (ThermoFisher) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM).

Techniques: Infection, Staining, Transfection, Expressing

Journal: bioRxiv

Article Title: Herpes simplex virus-1 pUL56 degrades GOPC to alter the plasma membrane proteome

doi: 10.1101/729343

Figure Lengend Snippet: HaCaT cells were infected at MOI of 10 in biological duplicate. Error bars: +/-standard deviation (SD). Plaque assays of HSV-1 WT and HSV-1 ΔUL56 in HaCaT, HFF hTERT, and Vero cells in biological duplicate. Cells were subsequently immunostained for the viral glycoprotein gD. C . Plaque diameters from ( B ) were measured and normalized to HSV-1 WT. Error bars: +/- SD, n = 35 to 67. D . Schematic of the proteomics workflow. Cells were infected at MOI of 10 or mock infected. E . Scatter plot of all proteins quantified, comparing HSV-1 WT and HSV-1 ΔUL56 at 8 hpi. Benjamini-Hochberg-corrected significance B was used to estimate p-values . F . Temporal profiles of all proteins downregulated >2-fold by HSV-1 WT vs mock and additionally rescued >2-fold by HSV-1 ΔUL56.

Article Snippet: HaCaT , telomerase immortalized human foreskin fibroblasts HFF hTERT , Vero (ATCC), HEK293T (ATCC), and Flp-In™-293 (ThermoFisher) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM).

Techniques: Infection, Standard Deviation