Article Figures & Data
Figures
- FIG 1
Schematic representation of two-step siRNA screen. Human fibroblast cells were transfected with siRNAs against 156 membrane-associated host genes (1) and infected with the GFP-expressing cytomegalovirus strain TB40/E GFP at 48 h posttransfection (2). Primary replication was tracked by measuring GFP expression. The supernatant, containing progeny virions, was transferred to untransfected cells, and the progress of infection was tracked by measuring GFP expression (3). A comparison of the primary replication at 4 dpi and virion progeny production at 4 dpi enriches for targets predominantly involved in the later stages of the viral life cycle.
- FIG 2
Primary replication (PR) and virus production (VP) are affected by siRNA knockdown of membrane-associated host genes. Relative primary replication of the virus based on GFP fluorescence from the first screen for each of the 156 knockdown experiments is shown in panel A, with the top five proviral and antiviral hits shown in panel B. Relative virus production based on GFP fluorescence from the second screen for each of the 156 knockdown experiments is shown in panel C, with the top five proviral and antiviral hits shown in panel D. All data were normalized to a scrambled siRNA control. n = 2; standard deviations shown. PR and VP measured 4 dpi.
- FIG 3
Primary replication is a poor predictor of virus production. Normalized GFP levels from primary replication (first screen, blue) and virus production (second screen, black) ranked by increasing primary replication show that a loss of primary replication does not necessarily lead to a loss of virus production (A). A direct comparison of normalized PR and VP levels was performed to determine correlation and therefore the predictive power of primary replication for virus production. Correlation was relatively low, with a Spearman score of 0.41 (B). This was not due to intrinsic variability of the screen, as correlation between repeats of PR, VP, and the ratio of PR to VP was high (see Fig. S1A, B, and C). Putative assembly and egress hits that show substantially reduced virus production with less than 2-fold effect on virus replication are highlighted in red and labeled, while antiviral hits that would not be identified following a conventional one-step screen with a 2-fold cutoff are shown in green (B). The top 10 putative assembly and egress hits are shown in panel C. n = 2; error bars reflect standard deviations. Two-tailed homoscedastic Student’s t test was applied to assess whether PR and VP results differed significantly. P > 0.05, NS; P ≤ 0.05, *; P ≤ 0.01,**; P ≤ 0.001, ***.
- FIG 4
Knockdown of putative assembly and egress hits result in substantial loss of virus replication. Fibroblast cells were transfected with pooled siRNAs against the indicated targets and infected 48 h posttransfection with TB40/E GFP at an MOI of 3. Supernatant and cells were harvested at the indicated times postinfection, and virus levels were determined by plaque assay (A and B). n = 2; error bars represent standard deviations.
- FIG 5
Inhibition of COPA, COPB2, ERC1, or RAB4B does not prevent virions from trafficking to the nucleus. Primary human fibroblast cells were transfected with indicated siRNAs and infected 48 h posttransfection with TB40/E GFP at MOIs of 0.1, 1, and 5. Cells were harvested 24 and 48 HPI, and GFP levels were measured by flow cytometry analysis to determine the effects on virus entry and genome translocation to the nucleus. Fold change in GFP-positive cells relative to cells transfected with a scrambled negative control is shown.
- FIG 6
Knockdown of COPA, COPB2, and ERC1, but not RAB4B, results in reduced viral DNA replication. Fibroblast cells were transfected with siRNA pools against indicated cellular targets or a control scrambled siRNA and infected with TB40/E GFP at an MOI of 3 at 48 h posttransfection. Total genomic DNA was isolated at the indicated time points, and viral genome levels were determined by qPCR. Inhibition of COPA and COPB2 resulted in a 7-fold and 4-fold loss of viral DNA replication by 7 DPI, respectively. A 2-fold loss of DNA replication was observed after siERC1 treatment. However, inhibition of RAB4B did not affect viral DNA replication. n = 2; error bars show standard deviations. *, P value < 0.05 determined by analysis of variance two-way test.
- FIG 7
Knockdown of COPA results in increase of pp28 protein expression. Fibroblast cells were transfected with siRNA pools against the indicated cellular targets (+) or a negative-control scrambled siRNA (−) and infected 48 h posttransfection with TB40/E GFP at an MOI of 3. Total protein was harvested, and levels of immediate early (IE1 and IE2), early (pp52), and late (pp28) proteins were monitored over 7 days postinfection (dpi) by Western blot analysis. ERC1 and COPB2 knockdown resulted in decreased levels of all three classes of viral protein (A to D). Knockdown of RAB4B resulted in an initial increase in IE protein expression. Expression of early and late proteins was not dramatically reduced (E and F). COPA inhibition did not affect IE or E proteins but led to a large increase in L protein levels (G and H).
- FIG 8
COPA inhibition results in increased late gene transcription. Fibroblast cells were transfected with siRNA pools against COPA, COPB2, or a negative-control scrambled siRNA and infected with TB40/E GFP 48 h posttransfection at an MOI of 3. Total RNA was harvested at the indicated time points, and relative viral transcript levels were determined by qRT-PCR for UL99 (pp28) (A), UL83 (pp65) (B), UL94 (C), and UL44 (pp52). Expression levels were normalized to GAPDH and expressed relative to the negative-control levels at the 7-day time point. n = 2; error bars show standard deviations. Two-tailed homoscedastic Student’s t test was applied to assess whether siCOPA treatment resulted in significantly different RNA levels compared to negative-control levels. P > 0.05, NS; P ≤ 0.05, *; P ≤ 0.01, **.
- FIG 9
Knockdown of RAB4B reduces levels of protected viral genomes in the supernatant. Fibroblast cells were transfected with RAB4B siRNA or a control scrambled siRNA and then infected 48 h posttransfection with HCMV at a high MOI. Supernatant was harvested 168 hpi, and viral particles were purified by ultracentrifugation. The virus particle was resuspended, and half the sample was treated with DNase before DNA isolation. Viral (A) (primers against gB) and cellular (B) (primers against GAPDH) DNA levels were then determined by qPCR. Data represent two biological repeats with error bars showing standard deviations. Two-tailed homoscedastic Student’s t test was applied to assess significance. P ≤ 0.05, *.
Supplemental Material
FIG S1
Correlation analysis between replicates of the screens shows a high level of reproducibility. Normalized GFP levels of biological repeats correlated well when comparing primary replication (A) (Spearman R, 0.87), virus production (B) (Spearman R, 0.94), and the ratio between virus production and primary replication (C) (Spearman R, 0.94). Download FIG S1, TIF file, 0.8 MB.
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FIG S2
Primary replication does not reliably predict virus production. Venn diagram showing the overlap between first screen, measuring primary replication, and second screen, measuring virus production. A total of 29 hits showed greater than 2-fold reduction in primary replication. Thirty-one hits showed greater than 2-fold reduction in virus production. Only 11 hits corresponded between the screens, showing a potentially high level of false-positive and -negative results using a conventional one-step screen that measures only virus replication. Download FIG S2, TIF file, 1.1 MB.
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TABLE S1
(A) Primary replication following siRNA treatment against 156 membrane-associated genes. Relative primary replication of TB40/E GFP in human fibroblast cells at 4 dpi is shown for each of 156 membrane-associated genes inhibited by siRNA treatment. GFP levels were normalized to a negative-control siRNA, and genes were ranked by decreasing effect on primary replication. n = 2; Std dev, standard deviations. (B) siRNA knockdown of membrane-associated genes resulted in the loss of virus production. The effect of siRNA treatment on primary replication and virus production relative to a negative control is shown, ranked by decreasing negative effect on virus production. n = 2; Std dev, standard deviations. Download TABLE S1, PDF file, 0.1 MB.
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TABLE S2
An increase in virus production ensued from siRNA knockdown of some membrane-associated genes. The fold change in primary replication and virus production relative to the negative control is shown, ranked by decreasing positive effect on virus production. n = 2; Std dev, standard deviations. Download TABLE S2, PDF file, 0.2 MB.
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TABLE S3
The ratio between virus production and primary replication following siRNA treatment against 156 membrane-associated genes. Genes were ranked by increasing VP/PR ratio to identify hits that had a larger effect on virus production than on primary replication. Two-tailed homoscedastic Student’s t test was applied to assess whether primary replication and virus production results differed significantly. P > 0.05, NS; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; P ≤ 0.0001, ****. Download TABLE S3, PDF file, 0.1 MB.
Copyright © 2018 McCormick et al.
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FIG S3
Confirmation of phenotypes using deconvoluted siRNAs. (A) Primary fibroblast cells were transfected with the 4 individual deconvoluted siRNAs for the top assembly and egress hits to rule out potential off-target effects. Total RNA was extracted 48 h posttransfection, and transcript levels were measured by qRT-PCR analysis to determine knockdown efficiency. In parallel, cells were infected with TB40/E GFP 48 h posttransfection, and GFP levels were measured using the two-step screen to determine primary replication and virus production. Knockdown efficiency was compared to effects on virus replication and virus production. (B) Western blot analysis showing ERC1 protein levels following knockdown with the pooled siRNA (P) or deconvoluted siRNAs A, B, C, and D. These were compared to cells transfected with a negative-control siRNA. (C) Quantification of protein levels compared to effects on primary replication and virus production. siRNAs B and D result in greater knockdown of ERC1 and a correspondingly higher reduction in virus production. (D) Fibroblast cells were transfected with pooled siRNAs against indicated cellular targets and cell viability was measured 48 h posttransfection using CellTiter-Blue. n = 2; error bars represent standard deviations. Download FIG S3, JPG file, 0.5 MB.
Copyright © 2018 McCormick et al.
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FIG S4
Inhibition of COPA, COPB2, ERC1 or RAB4B does not prevent virions from trafficking to the nucleus. Primary human fibroblast cells were transfected with indicated siRNAs and infected 48 h posttransfection with TB40/E GFP at MOIs of 0.1, 1 and 5. Cells were harvested 24 and 48 HPI and GFP levels were measured by flow cytometry analysis to determine the effects on virus entry and genome translocation to the nucleus. Fluorescence is shown as MFI. Download FIG S4, JPG file, 0.9 MB.
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FIG S5
siRNA knockdown of COPA, COPB2, ERC1, or RAB4B does not prevent the translocation of TB40/E GFP DNA to the nucleus. Fluorescence microscopy analysis of human fibroblast cells transfected with siRNAs against COPA, COPB2, RAB4B, ERC1, or a negative-control siRNA. Cells were infected at an MOI of 3 with TB40/E GFP and imaged at 1 and 2 dpi. Download FIG S5, JPG file, 2.3 MB.
Copyright © 2018 McCormick et al.
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FIG S6
Correlation of reduced COPA mRNA levels with increased pp28 mRNA levels. Primary fibroblast cells were transfected with 4 individual COPA deconvoluted siRNAs, a pool of COPA siRNAs, or a negative-control siRNA and infected 48 h posttransfection with TB40/E GFP at an MOI of 3. Total RNA was extracted 6 dpi, and transcript levels were measured by qRT-PCR analysis. The normalized levels of pp28 RNA were found to increase with a corresponding decrease in the normalized levels of COPA RNA (Spearman R, 0.68). Download FIG S6, JPG file, 0.3 MB.
Copyright © 2018 McCormick et al.
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FIG S7
RAB4B relocates to the VAC at late stages of HCMV infection. Fibroblast cells were infected with HCMV and fixed 120 hpi. Cells were stained for the viral tegument protein pp28 (red), which colocalizes with the VAC, RAB4B (green), and DAPI (blue). Punctate staining throughout the cytoplasm and particularly around the nucleus can be seen in uninfected cells. By 120 hpi, RAB4B staining relocates to the VAC, colocalizing with the viral tegument protein pp28. Download FIG S7, JPG file, 2.5 MB.
Copyright © 2018 McCormick et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
