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

Wu, Kai; Oberstein, Adam; Wang, Wei; Shenk, Thomas

doi:10.1073/pnas.1806305115

Cited by 81 publications

(105 citation statements)

References 52 publications

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“…Multiple reports have monitored foci size in the presence of neutralizing antibodies and attributed the cell-associated nature of ME to the expression of the UL128-131 and RL13 loci (24, 42). It seems clear that either gH/gL/gO or gH/gL/UL128-131 is required for cell-associated spread on fibroblasts, whereas gH/gL/UL128-131 seems to be required for cell-associated spread in epithelial or endothelial cells (24, 26, 43, 44). Moreover, the pathway of cell-associated spread for HCMV has not been extensively studied.…”

Section: Introductionmentioning

confidence: 99%

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Schultz

Lanchy

Day

et al. 2019

Preprint

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ABSTRACTIt is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell-associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The BAC-clone Merlin (ME), expresses abundant UL128-131, is RL13-impaired and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, RL13-intact and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread, but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptional repressed, cell-to-cell spread of ME was still more efficient than TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci.IMPORTANCEBoth cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether “cell-associated” phenotypes are simply the result of poor cell-free spread, or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

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Section: Introductionmentioning

confidence: 99%

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Schultz

Lanchy

Day

et al. 2019

Preprint

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“…Recent studies have shown that PDGFRα specifically interacts with the gO subunit of the trimer which is required for entry into fibroblasts (25–28). As soluble forms of PDGFRα or derivatives thereof can inhibit cell-free infection of several cell types (26) it appears that binding of sPDGFRα to gO interferes with trimer-mediated function(s) widely required for cell entry.…”

Section: Discussionmentioning

confidence: 99%

“…One of these cellular receptors, platelet-derived growth factor receptor alpha (PDGFRα), was identified to directly and specifically interact with gO parts of the trimer (25–27). This interaction enables entry of cell-free virions into fibroblasts, the only cell type which shows a high PDGFRα expression (28). Albeit, soluble forms of PDGFRα (sPDGFRα) can severely inhibit not only entry into fibroblasts, but also entry into endothelial and epithelial cells (25–27), and first observations indicate that the inhibitory capacity of sPDGFRα is effective against several HCMV strains even when they harbour a different gO genotype sequence (26).…”

Section: Introductionmentioning

confidence: 99%

Influence of human cytomegalovirus glycoprotein O polymorphism on the inhibitory effect of soluble forms of trimer- and pentamer-specific entry receptors

Brait

Stögerer

Kalser

et al. 2019

Preprint

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AbstractHuman cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO-trimer and gH/gL/UL128L-pentamer, are important for cell-free HCMV entry. While soluble Nrp2-Fc (sNrp2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble PDGFRα-Fc (sPDGFRα-Fc) interacts with gO thereby inhibiting infection of all cell types. Since gO is the most variable subunit we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc.Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, GT5 sequences in TB40-BAC4-luc background. All mutants were tested for fibroblast and epithelial cell infectivity, for virions’ gO and gH content, and for infection inhibition by sPDGFRα-Fc and sNrp2-Fc.Full-length and partial gO GT swapping may strongly alter the virions’ gO and gH levels associated with enhanced epithelial cell infectivity. All gO GT mutants except recombinant gO GT1c/3 displayed a near-complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%) and all on fibroblasts (≥ 99%). While gO GT replacement did not influence sNrp2-Fc inhibition at 1.25 μg/ml on epithelial cells (96%-98%), it rendered mutants with low gO levels moderately accessible to fibroblasts inhibition (20%-40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope >1.0), sNrp2-Fc dose-response curves on epithelial cells displayed slopes of ~1.0 suggesting functional differences between these entry inhibitors.Our findings suggest that targeting of gO-trimer rather than UL128-pentamer might be a promising target to inhibit infectivity independent of the cell type, gO polymorphism, and gO/gH content. However, intragenic gO recombination may lead to moderate resistence to sPDGFRα-Fc inhibition.ImportanceHuman cytomegalovirus (HCMV) is known for its broad cell tropism as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered as a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the virions’ amount of trimer and the highly polymorphic gO sequence. In this study, we show that gO polymorphism rather than gO levels may affect the inhibitory capacity of sPDGFRα. The finding that gO intragenic recombination may lead to moderate evasion from sPDGFRα inhibition is of major value to the development of potential anti-HCMV therapeutic compounds based on sPDGFRα.

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“…In brief, UL148 appears to stabilize immature forms of glycoprotein O (gO) within the ER, thereby promoting the maturation and incorporation into virions of the heterotrimeric glycoprotein H (gH)/ glycoprotein L (gL)/ gO complex. Since gH/gL/gO is one of the two alternative gH/gL complexes that endow virus with the ability to utilize specific host cell surface receptors during entry, in this case with gO being required to achieve cell entry via PDGFR α , which is well expressed in fibroblasts but poorly expressed in epithelial cells (19–22), the effects of UL148 on trimer expression are thought to account for the ∼100-fold improved replication of UL148 -null viruses in epithelial cells (17, 18).…”

Section: Introductionmentioning

confidence: 99%

“…gH/gL/gO (Trimer) and gH/gL/UL128-131 (Pentamer) are the two alternative HCMV gH/gL complexes that endow the virus with the ability to utilize different proteinaceous host cell surface factors as entry receptors [reviewed in (19)]. In particular, gO, in the context of Trimer, is required for viral entry via the platelet-derived growth factor receptor alpha (PDGFR α ), which is well expressed in fibroblasts but poorly if at all in epithelial cells (20–23). Infection of epithelial and endothelial cells, on the other hand, appears to require direct interactions between the Pentamer and neuropilin-2 (Nrp2) (24), together with roles for OR14I1 (25) and CD147 (26), addition to roles for the Trimer (27, 28).…”

Section: Introductionmentioning

confidence: 99%

ER reorganization and intracellular retention of CD58 are functionally independent properties of the human cytomegalovirus ER resident glycoprotein UL148

Nguyen

Zhang

et al. 2019

Preprint

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The human cytomegalovirus (HCMV) endoplasmic reticulum (ER)-resident glycoprotein UL148 is posited to play roles in immune evasion and regulation of viral cell tropism. UL148 prevents cell surface presentation of the immune cell costimulatory ligand CD58 while promoting maturation and virion incorporation of glycoprotein O, a receptor binding subunit for an envelope glycoprotein complex involved in entry. Meanwhile, UL148 activates the unfolded protein response (UPR) and causes large-scale reorganization of the ER. In an effort to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated charged-cluster-to-alanine (CCTA) mutants of six charged clusters within the UL148 ectodomain, and compared them against wildtype UL148, in the context of recombinant viruses and in ectopic expression, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79-83 (CC3) and 133-136 (CC4), respectively, retained the potential to impede CD58 presentation, and did so to an extent comparable to wildtype. Of the six mutants, only CC3 retained the capacity to reorganize the ER, showing a partial phenotype. Wildtype UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which may imply a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses CD58 presentation but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate varying degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that ER reorganization is unique to HCMV UL148.IMPORTANCEIn myriad examples, viral gene products cause striking effects on cells, such as activation of stress responses. It can be challenging to decipher how such effects contribute to the biological roles of the proteins. The HCMV glycoprotein UL148 retains CD58 within the ER, thereby preventing it from reaching the cell surface where it functions to stimulate cell-mediated antiviral responses. Intriguingly, UL148 also triggers the formation of large, ER-derived membranous structures, and activates the UPR, a set of signaling pathways involved in adaptation to ER stress. We demonstrate that the potential of UL148 to reorganize the ER and to retain CD58 are separable by mutagenesis and possibly, by evolution, since chimpanzee cytomegalovirus UL148 retains CD58 but does not remodel the ER. Our findings imply that ER reorganization contributes to other roles of UL148, such as modulation of alternative viral glycoprotein complexes that govern the virus’ ability to infect different cell types.

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

Cited by 81 publications

References 52 publications

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

Influence of human cytomegalovirus glycoprotein O polymorphism on the inhibitory effect of soluble forms of trimer- and pentamer-specific entry receptors

ER reorganization and intracellular retention of CD58 are functionally independent properties of the human cytomegalovirus ER resident glycoprotein UL148

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