Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection - PubMed (original) (raw)

Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection

Daniel Hochdorfer et al. J Virol. 2016.

Abstract

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins that mediate binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown. In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells by use of a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five proviral host factors from different protein families and eight antiviral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel proviral host factor and was analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either broad or narrow cell tropism were equally impaired. Infection of CD151-depleted cells by a fluorescent virus with differentially labeled capsid and envelope proteins revealed a role of CD151 in viral penetration but not in adsorption to the cell. In conclusion, the tetraspanin CD151 has emerged as a novel host factor in HCMV entry and as a putative antiviral target.

Importance: At present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with putative roles in this process were identified. The tetraspanin CD151 was discovered as a previously unrecognized proviral host factor for HCMV and was found to support viral penetration into the target cells. The findings of this study shed light on the cellular contribution during the initial steps of HCMV infection and open a new direction in HCMV research.

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Figures

FIG 1

Identification of host factors in initial steps of HCMV infection by RNAi screen. (A) A fluorescently labeled siRNA (red) was transfected into EA.hy926 cells, and uptake was monitored at 1 day (d) posttransfection. The cells were subsequently infected with HCMV strain TB40/E at 48 h posttransfection, and infected cells were visualized at 1 day p.i. by immunostaining of viral immediate early antigens (IE) pUL122 and pUL123 (green). Nuclei were stained with DAPI (blue). (B) Metabolic conversion of resazurin to the fluorescent molecule resorufin served as an indicator of cell viability. EA.hy926 cells were transfected with a nontargeting pool (NT) or RISC-free (RF) siRNA and compared to untransfected cells. Raw data for duplicates from four experiments (n = 8) were standardized based on the untransfected cells. (C) In situ ELISA was employed to measure immediate early antigen (IE-Ag) levels in infected cells. Serial virus dilutions were prepared and used to infect EA.hy926 cells. In order to test whether the ELISA results could serve as an indirect readout for the fraction of infected EA.hy926 cells determined by IE-Ag detection via immunofluorescence, both readouts were performed in parallel. The ELISA data (black) were plotted with the percentages of infected cells (red) in the same graph to show the degree of correlation. (D) Schematic overview of the RNAi screening strategy. Four individual experiments (gray) were carried out with an RNAi library encompassing 96 human target genes. The data from all experiments (two replicates per experiment) were analyzed collectively to determine “hits.” (E) Ranking of the targeted genes according to their corresponding ELISA results for hit identification. In both panels E and F, the median for all samples is shown as a solid gray line, and the cutoffs (median ± 2 standard deviations [for the negative controls, i.e., untransfected cells, NT siRNA-transfected cells, and RF siRNA-transfected cells]) are shown as dashed red lines. (F) The screen results for the four tetraspanin genes covered by the library (CD9, CD63, CD81, and CD151) are shown and compared to the mean for the negative controls (untransfected cells, NT siRNA-transfected cells, and RF siRNA-transfected cells). Error bars show standard errors of the means. n.s., not significant; *, significant (P < 0.05; Student's t test); rel. OD492, relative optical density at 492 nm.

FIG 2

Regulation by CD151 siRNA was efficient and specific. (A) CD151 protein levels were analyzed by Western blot analysis of EA.hy926 cells 48 h after transfection with CD151 or nontargeting (NT) siRNA. A nonspecific band produced by the CD151 antibody served as a loading control. (B) Live EA.hy926 cells were immunostained for CD151 using a Cy3-conjugated secondary antibody (red) after siRNA treatment (NT or CD151 siRNA). After fixation, nuclei were counterstained with DAPI (blue). (C) The indicated siRNAs were transiently cotransfected into EA.hy926 cells with a plasmid conferring immediate early antigen (IE-Ag) expression in the absence of infection. Error bars show standard errors of the means. n.s., not significant; *, significant (P < 0.05; Student's t test).

FIG 3

CD151 siRNAs specifically reduced the infection of EA.hy926 cells. (A to D) Individual CD151 siRNAs from the siRNA pool used in the screen or an equimolar mixture thereof were transfected 48 h prior to infection. Immediate early antigen (IE-Ag) expression was measured by ELISA (A), and the fraction of infected cells was determined by indirect immunofluorescence staining of IE-Ag (B and D) with a primary antibody and a Cy3-conjugated secondary antibody (red). Nuclei were counterstained with DAPI (blue). (C) Magnified images showing pan-nuclear (NT, IE, and CD151 siRNAs) or punctate (IE siRNA) (white arrowhead) expression pattern of IE-Ag. siRNAs: NT, nontargeting pool; IE, immediate early antigen; CDS, CD151 coding sequence; 3′ UTR, CD151 3′ untranslated region; pool, equimolar mix of both CD151 siRNAs. (E) A CD151 siRNA that targets the 3′ UTR of the CD151 gene was cotransfected with a plasmid encoding either GFP (as a control) or a CD151-GFP transgene resistant to the siRNA. NT siRNA was used as a reference, and IE siRNA was used as a positive control. The data were standardized to the negative control (NT siRNA plus GFP plasmid). The graph shows mean values for three independent experiments. Error bars show standard errors of the means. n.s., not significant; *, significant (P < 0.05; Student's t test).

FIG 4

The contribution of CD151 to HCMV infection was not cell type restricted. (A and B) Relative fractions of HCMV-infected HEC-LTTs (A) or HFFs (B) as measured by immunofluorescence staining of immediate early antigens. Cells were transfected with a pool of CD151 siRNAs or control siRNAs, as indicated, 48 h prior to infection. NT siRNA-treated cells served as a reference, and IE siRNA was used as a positive control. (A) HEC-LTTs were infected with HCMV strain TB40/E. (B) HFFs were infected with a highly endotheliotropic (TB40/E) or poorly endotheliotropic (TB40/F) strain. Error bars show standard errors of the means. *, significant (P < 0.05; Student's t test).

FIG 5

CD151 depletion affected viral penetration but not adsorption. (A) Exemplary images showing fluorescence signals in a cell infected with the dual-fluorescence virus strain TB40-BACKL7-UL32eGFP-UL100mCherry. Green signals were interpreted as EGFP-labeled capsids (green arrowheads) and red signals as mCherry-labeled viral envelopes. In the merged image, particles showing both green and red signals appear yellow (yellow arrowhead). The cell nucleus was counterstained with DAPI (blue). (B and C) Fibroblasts (HFFs) and endothelial cells (HEC-LTTs) were transfected for 48 h with CD151 or NT siRNA and then inoculated with the dual-fluorescence HCMV strain TB40-BACKL7-UL32eGFP-UL100mCherry at an MOI of 20 to 80. Cells were fixed after 30 min. Fluorescence images were taken and analyzed by determining the total number of green signals (all capsids, regardless of the presence or absence of envelope) (B) and the fraction of green-only signals, without red (nonenveloped particles) (C), to determine the total number of adsorbed virus particles (B) and the fraction of successfully penetrated particles (C). In each experiment, >800 particles were counted for each condition. The penetration efficiency was standardized to the NT control level. Error bars show standard errors of the means. n.s., not significant; *, significant (P < 0.05; Student's t test).

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Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection - PubMed (original) (raw)