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. 2018 Oct 16;115(42):E9889-E9898.
doi: 10.1073/pnas.1806305115. Epub 2018 Oct 1.

Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

Kai Wu  1 , Adam Oberstein  1 , Wei Wang  2 , Thomas Shenk  3
Affiliations

Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

Kai Wu et al. Proc Natl Acad Sci U S A. .

Abstract

Human CMV (HCMV) exhibits a broad cell tropism that depends on two virion glycoprotein complexes: a trimeric complex (gH/gL/gO) that facilitates viral infection primarily in fibroblasts and a pentameric complex (gH/gL/pUL128-pUL130-pUL131A) that mediates infection in epithelial and endothelial cells. We performed genome-wide CRISPR screens in which the PDGF receptor-α (PDGFRα) was identified as the most significant cellular gene product essential for infection by HCMV virions containing only trimeric complex (trimer-only virus). Trimer-only virus did not enter PDGFRα knockout fibroblasts. By using knockout fibroblasts, the extracellular domain of PDGFRα required for virus entry was mapped, and the intracellular tyrosine kinase domain was shown to be nonessential. In addition, direct cell-to-cell spread of virus from knockout cells transfected with trimer-only viral DNA was blocked, despite the production of infectious virus in the transfected cells. In contrast to trimer-only virus, wild-type HCMV virions containing both trimeric and pentameric complexes entered PDGFRα knockout cells, reinforcing the view that fibroblasts contain a second, independent receptor for the pentameric complex. Importantly, however, wild-type virus entered the knockout fibroblasts at reduced efficiency compared with parental fibroblasts, arguing that the cellular receptor for the virion pentameric complex is limiting or that virions are produced containing different relative amounts of the two glycoprotein complexes. Finally, ectopic expression of PDGFRα in ARPE-19 epithelial cells and THP-1 monocytic cells, which have little to no endogenous PDGFRα expression, markedly enhanced their susceptibility to trimer-only virions. In sum, our data clarify several key determinants of HCMV tropism.

Keywords: cellular receptor; herpesvirus; tropism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HFFs expressing Cas9 and gRNAs targeting PDGFRα survived trimer-only HCMV infection in a genome-wide CRISPR screen. Statistical analysis identified PDGFRα as the most significant hit from the screens of AD169 (A) and Merlin (B), using the MAGeCK algorithm.
Fig. 2.
Fig. 2.
Trimer-only HCMV cannot initiate infection in PDGFRα-KO HFF cells. (A) Single-cell cloning of HFFs transduced with lentivirus expressing Cas9 and gRNAs against PDGFRα generated three PDGFRα-KO clones from three independent gRNAs. HFFs (CN-9) that underwent the same single-cell cloning process served as an aged control. Surface staining of PDGFRα and PDGFRβ was performed to confirm the PDGFRα-KO phenotype. (B) Cells in A were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and cell morphology was monitored at 5 dpi. (C) Cell-free virus production at 5 dpi was measured by plaque assay. Mean ± SD is presented from three biological replicates, and the red line indicates the limit of detection.
Fig. 3.
Fig. 3.
IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. (A) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. (B and C) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. (D) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.
Fig. 4.
Fig. 4.
PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. (A) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. (B) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. (C) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.
Fig. 5.
Fig. 5.
Virus containing pentamer enters PDGFRα-KO HFF cells using an alternative route(s). HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with viruses produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells, except for Merlin-GFP-epi and Merlin-GFP-fibro, where IE1 expression was measured to assay percentage of infected cells due to the dim GFP signal. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively.
Fig. 6.
Fig. 6.
Pentamer-containing TB40/E raised from ARPE-19 epithelial cells enters PDGFRα-KO HFFs more efficiently than TB40/E grown from fibroblasts. (A) HFFs, PDGFRα-KO cell clones (1-10 and 10-4), and CN-9 control cells were infected with TB40/E-GFP produced from fibroblasts or epithelial cells at a multiplicity of 3 FFU per cell. At 2 dpi, GFP expression from viral genomes was measured by flow cytometry to assay the percentage of infected cells. The suffixes -epi and -fibro designate virus stocks produced in ARPE-19 epithelial and HFF fibroblast cells, respectively. (B) Western blot analysis of virions produced from MRC-5 cells infected by TB40/E-fibro or TB40/E-epi for gH/gL complexes (nonreducing conditions) using anti-gH antibody and for viral major capsid protein (MCP; reducing conditions) serving as loading control for virion proteins.
Fig. 7.
Fig. 7.
Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 (A) or THP-1 (B) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. (Top) Cells were subjected to flow cytometry using the indicated antibodies. (Bottom) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.
Fig. 8.
Fig. 8.
Virus containing the pentameric complex produced from epithelial cells efficiently enters primary human monocytes. CD14+ monocytes were purified from three donors and cell-surface staining of CD14 (A) or PDGFRα and PDGFRβ (B) were analyzed before culture in medium containing autologous serum. Purified CD14+ monocytes were infected with Merlin-epi (3 FFU per cell) or Merlin-fi (10 and 3 FFU/cell) for 16 h and intracellular staining of IE1 was performed to assay the percentage of infected cells (C).
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