CCR5 adopts three homodimeric conformations that control cell surface delivery
2018; 11 (529)
Biophysical methods and x-ray crystallography have revealed that class A G protein-coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition.
View details for DOI 10.1126/scisignal.aal2869
View details for Web of Science ID 000431764800001
View details for PubMedID 29739880
Transcriptome analysis reveals dynamic changes in coxsackievirus A16 infected HEK 293T cells
BIOMED CENTRAL LTD. 2017: 933
Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are two of the major causes of hand, foot and mouth disease (HFMD) world-wide. Although many studies have focused on infection and pathogenic mechanisms, the transcriptome profile of the host cell upon CVA16 infection is still largely unknown.In this study, we compared the mRNA and miRNA expression profiles of human embryonic kidney 293T cells infected and non-infected with CVA16. We highlighted that the transcription of SCARB2, a cellular receptor for both CVA16 and EV71, was up-regulated by nearly 10-fold in infected cells compared to non-infected cells. The up-regulation of SCARB2 transcription induced by CVA16 may increase the possibility of subsequent infection of CVA16/EV71, resulting in the co-infection with two viruses in a single cell. This explanation would partly account for the co-circulation and genetic recombination of a great number of EV71 and CVA16 viruses. Based on correlation analysis of miRNAs and genes, we speculated that the high expression of SCARB2 is modulated by down-regulation of miRNA has-miR-3605-5p. At the same time, we found that differentially expressed miRNA target genes were mainly reflected in the extracellular membrane (ECM)-receptor interaction and circadian rhythm pathways, which may be related to clinical symptoms of patients infected with CVA16, such as aphthous ulcers, cough, myocarditis, somnolence and potentially meningoencephalitis. The miRNAs hsa-miR-149-3p and hsa-miR-5001-5p may result in up-regulation of genes in these morbigenous pathways related to CVA16 and further cause clinical symptoms.The present study elucidated the changes in 293T cells upon CVA16 infection at transcriptome level, containing highly up-regulated SCARB2 and genes in ECM-receptor interaction and circadian rhythm pathways, and key miRNAs in gene expression regulation. These results provided novel insight into the pathogenesis of HFMD induced by CVA16 infection.
View details for DOI 10.1186/s12864-016-3253-6
View details for Web of Science ID 000408766800002
View details for PubMedID 28198671
View details for PubMedCentralID PMC5310284
Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (27): 19042-19052
CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.
View details for DOI 10.1074/jbc.M114.559831
View details for Web of Science ID 000339062900035
View details for PubMedID 24855645
View details for PubMedCentralID PMC4081942
Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum
JOURNAL OF VIROLOGICAL METHODS
2013; 187 (2): 362–67
Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.
View details for DOI 10.1016/j.jviromet.2012.11.014
View details for Web of Science ID 000315074900026
View details for PubMedID 23178532