Journal: The Journal of General Virology

Article Title: HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L

doi: 10.1099/jgv.0.001980

Figure Lengend Snippet: US2 degrades LMAN2L via the proteasome. (a) Venn diagram showing the overlap between shortlists of proteins: (i) degraded early during HCMV infection ; (ii) significantly rescued upon infection with an HCMV gene-block deletion mutant compared to wild-type infection [55]; and (iii) assigned the gene ontology term ‘protein transport’ (GO:0015031) according to the AmiGO database [ , ]. The following two panels (b, c) are based on data from our prior publication [55], and are the only previously published data in this paper. (b) Three orthogonal protein degradation screens showing that LMAN2L is degraded with medium confidence during early HCMV infection (from [55]). Left panel: LMAN2L was rescued from degradation by the proteasome inhibitor MG132 in HCMV-infected cells, but not during mock infection, infection with UV-irradiated HCMV (HCMV*) or inhibition with the lysosomal protease inhibitor leupeptin (see also figure 1 from [55]). Middle panel: increased rate of LMAN2L degradation from 4 h of HCMV compared to mock infection. Cells were pre-labelled with medium SILAC amino acids prior to infection, then switched to heavy amino acids at the point of infection. Quantification of medium-labelled proteins over time using tandem mass tag (TMT)-based multiplexing facilitated quantification of protein degradation rates (see also figure 2 from [55]). Right panel: relative abundance of LMAN2L protein and transcript over 72 h of infection. (c) A proteomic screen of HCMV block deletion mutants determined that the US1–US11 gene block is required to downregulate LMAN2L [55]. Abundance is shown relative to WT HCMV. A value of 1 indicates no change. (d) Immunoblot showing expression of endogenous LMAN2L in HFFF-CAR infected with RAd expressing V5-tagged US1–US11 genes (m.o.i. 10, 72 h). LMAN2L abundance was normalized to GAPDH and is shown relative to the control RAd given a value of 1. Error bars represent the range ( n =2). Validation of transgene expression is shown in Fig. S1. (e) Immunoblot of lysates from HFFF-TERTs infected with strain Merlin HCMV, and ΔUS2, ΔUS3 or ΔUS11 recombinants (m.o.i. 5, 24 h), or mock infected. Immunoblot shown is representative of two independent experiments.

Article Snippet: Low passage HFFF-TERTs were nucleofected with ribonucleoprotein (RNP) complexes containing recombinant Cas9 nuclease (IDT, 1081059) and guide RNAs (gRNA) against LMAN2L or a non-targeting negative control (Synthego), using the Nucleofector 2d Transfection Device (Lonza).

Techniques: Infection, Blocking Assay, Mutagenesis, Irradiation, Inhibition, Protease Inhibitor, Multiplex sample analysis, Multiplexing, Western Blot, Expressing, Control, Biomarker Discovery

Journal: The Journal of General Virology

Article Title: HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L

doi: 10.1099/jgv.0.001980

Figure Lengend Snippet: Cellular E3 ligase TRC8 is necessary for LMAN2L degradation. ( a ) Schematic of the mechanism of US2 and TRC8-mediated degradation of membrane proteins, adapted from Hsu et al . . ( b ) Confirmation of TRC8 knockdown. HFFF-TERTs were transduced with TRC8 or scrambled control shRNA plasmids and knockdown efficiency was quantified by RT-qPCR. Relative RNA levels were calculated using the 2 −ΔΔCT method . ( c ) LMAN2L expression in HCMV-infected TRC8 shRNA knockdown cells. Control (-) and TRC8 shRNA (+) cell lines were infected with strain Merlin WT HCMV or a ΔUS2 strain Merlin recombinant for 24 h (m.o.i. 5), and LMAN2L expression was measured by immunoblot. The LMAN2L signal was normalized to GAPDH and is shown relative to mock control samples. The average relative abundance is shown across three biological replicates and error bars represent the standard error of the mean, except for samples infected with ΔUS2 where there were no biological replicates. P -values were calculated using a two-sample unpaired t-test. * P <0.05.

Article Snippet: Low passage HFFF-TERTs were nucleofected with ribonucleoprotein (RNP) complexes containing recombinant Cas9 nuclease (IDT, 1081059) and guide RNAs (gRNA) against LMAN2L or a non-targeting negative control (Synthego), using the Nucleofector 2d Transfection Device (Lonza).

Techniques: Membrane, Knockdown, Transduction, Control, shRNA, Quantitative RT-PCR, Expressing, Infection, Recombinant, Western Blot

Journal: The Journal of General Virology

Article Title: HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L

doi: 10.1099/jgv.0.001980

Figure Lengend Snippet: LMAN2L-dependent expression of proteins at the plasma membrane. ( a ) Schematic of the PMP workflow. ( b ) Immunoblot validation of LMAN2L knockdown/knockout in bulk CRISPR KO cells (top) and after siRNA treatment (bottom). Percentage decrease was calculated relative to the corresponding negative controls (ctrl). ( c ) Scatterplot comparing the average fold change from siRNA ( n =3) and CRISPR samples ( n =1). A coefficient of variation (CV) was calculated by dividing the standard deviation by the average fold change across siRNA and bulk CRISPR samples and multiplying by 100. Entries with a CV>30 were excluded from further analysis. Significance A was used to calculate P -values, which were corrected for multiple testing using the Benjamini–Hochberg method [ ]. Proteins that were significantly downregulated ( P <0.05) in both LMAN2L siRNA and LMAN2L bulk KO are labelled and highlighted in orange. ( d ) Fold change of proteins significantly downregulated in both LMAN2L siRNA knockdown and bulk LMAN2L KO cells compared to the corresponding control. Error bars represent the standard error of the mean. ( e ) Fold change of all integrin molecules quantified in the proteomic screen. The bottom and top of the box show the first and third quartile, respectively, and the median is shown as the line inside the box. Individual fold changes for the replicates are shown as data points.

Article Snippet: Low passage HFFF-TERTs were nucleofected with ribonucleoprotein (RNP) complexes containing recombinant Cas9 nuclease (IDT, 1081059) and guide RNAs (gRNA) against LMAN2L or a non-targeting negative control (Synthego), using the Nucleofector 2d Transfection Device (Lonza).

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot, Biomarker Discovery, Knockdown, Knock-Out, CRISPR, Standard Deviation, Control