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Role of PDGF receptor-? during human cytomegalovirus entry into fibroblasts.

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-? (PDGFR?) 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 PDGFR? knockout fibroblasts. By using knockout fibroblasts, the extracellular domain of PDGFR? 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 PDGFR? 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 PDGFR? in ARPE-19 epithelial cells and THP-1 monocytic cells, which have little to no endogenous PDGFR? expression, markedly enhanced their susceptibility to trimer-only virions. In sum, our data clarify several key determinants of HCMV tropism.

SUBMITTER: Wu K

PROVIDER: S-EPMC6196492 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Wu Kai K Oberstein Adam A Wang Wei W Shenk Thomas T

Proceedings of the National Academy of Sciences of the United States of America 20181001 42

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 tri ...[more]

PMID: 30275317

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Project description:A vaccine to prevent congenital cytomegalovirus (CMV) infections is a national priority. Investigational vaccines have targeted the viral glycoprotein B (gB) as an inducer of neutralizing antibodies and phosphoprotein 65 (pp65) as an inducer of cytotoxic T cells. Antibodies to gB neutralize CMV entry into all cell types but their potency is low compared to antibodies that block epithelial cell entry through targeting the pentameric complex (gH/gL/UL128/UL130/UL131). Hence, more potent overall neutralizing responses may result from a vaccine that combines gB with pentameric complex-derived antigens. To assess the ability of pentameric complex subunits to generate epithelial entry neutralizing antibodies, DNA vaccines encoding UL128, UL130, and/or UL131 were formulated with Vaxfectin(®), an adjuvant that enhances antibody responses to DNA vaccines. Mice were immunized with individual DNA vaccines or with pair-wise or trivalent combinations. Only the UL130 vaccine induced epithelial entry neutralizing antibodies and no synergy was observed from bi- or trivalent combinations. In rabbits the UL130 vaccine again induced epithelial entry neutralizing antibodies while UL128 or UL131 vaccines did not. To evaluate compatibility of the UL130 vaccine with DNA vaccines encoding gB or pp65, mono-, bi-, or trivalent combinations were evaluated. Fibroblast and epithelial entry neutralizing titers did not differ between rabbits immunized with gB alone vs. gB/UL130, gB/pp65, or gB/UL130/pp65 combinations, indicating a lack of antagonism from coadministration of DNA vaccines. Importantly, gB-induced epithelial entry neutralizing titers were substantially higher than activities induced by UL130, and both fibroblast and epithelial entry neutralizing titers induced by gB alone as well as gB/pp65 or gB/UL130/pp65 combinations were comparable to those observed in sera from humans with naturally-acquired CMV infections. These findings support further development of Vaxfectin(®)-formulated gB-expressing DNA vaccine for prevention of congenital CMV infections.

Dataset Information

Role of PDGF receptor-? during human cytomegalovirus entry into fibroblasts.

Publications

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

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