Dataset Information

Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus.

ABSTRACT: JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by ?-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of ?-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.

SUBMITTER: Assetta B

PROVIDER: S-EPMC6544161 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus.

Assetta Benedetta B Morris-Love Jenna J Gee Gretchen V GV Atkinson Abigail L AL O'Hara Bethany A BA Maginnis Melissa S MS Haley Sheila A SA Atwood Walter J WJ

Cell reports 20190501 7

JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISP ...[more]

PMID: 31091436

Similar Datasets

Project description:JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People that become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or significant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins β-arrestin, adaptor protein 2 (AP2), and dynamin. Further, a β-arrestin interacting domain, the Ala-Ser-Lys (ASK) motif, within the C-terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5-HT2R ASK motifs and the activation of β-arrestin-associated proteins during internalization has not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as critical for JCPyV internalization and infectivity. Further, utilizing biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced β-arrestin binding. Utilizing small-molecule chemical inhibitors and RNA interference, G-protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between β-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and β-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and resultant infection.IMPORTANCE As intracellular parasites, viruses require a host cell to replicate and cause disease. Therefore, virus-host interactions contribute to viral pathogenesis. JC polyomavirus (JCPyV) infects most of the population, establishing a lifelong asymptomatic infection within the kidney. Under conditions of severe immunosuppression JCPyV may spread to the central nervous system, causing the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals living with HIV or undergoing immunomodulatory therapies are at risk for developing PML. The mechanisms of how JCPyV uses specific receptors on the surface of host cells to initiate internalization and infection is a poorly understood process. We have further identified cellular proteins involved in JCPyV internalization and infection and elucidated their specific interactions that are responsible for activation of receptors. Collectively, these findings illuminate how viruses usurp cellular receptors during infection, contributing to current development efforts for therapeutic options for the treatment or prevention of PML.

Project description:JC polyomavirus (JCPyV) establishes a persistent, lifelong, asymptomatic infection within the kidney of the majority of the human population. Under conditions of severe immunosuppression or immune modulation, JCPyV can reactivate in the central nervous system (CNS) and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. Initiation of infection is mediated through viral attachment to α2,6-sialic acid-containing lactoseries tetrasaccharide c (LSTc) on the surface of host cells. JCPyV internalization is dependent on serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs), and entry is thought to occur by clathrin-mediated endocytosis (CME). However, the JCPyV entry process and the cellular factors involved in viral internalization remain poorly understood. Treatment of cells with small-molecule chemical inhibitors and RNA interference of 5-HT2R endocytic machinery, including β-arrestin, clathrin, AP2, and dynamin, significantly reduced JCPyV infection. However, infectivity of the polyomavirus simian virus 40 (SV40) was not affected by CME-specific treatments. Inhibition of clathrin or β-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a β-arrestin binding domain (Ala-Ser-Lys) within the intracellular C terminus of 5-HT2AR severely diminished internalization and infection, suggesting that β-arrestin interactions with 5-HT2AR are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrin-mediated endocytosis of JCPyV, which is necessary for viral internalization and productive infection.IMPORTANCE Viruses usurp cellular factors to invade host cells. Activation and utilization of these proteins upon initiation of viral infection are therefore required for productive infection and resultant viral disease. The majority of healthy individuals are asymptomatically infected by JC polyomavirus (JCPyV), but if the host immune system is compromised, JCPyV can cause progressive multifocal leukoencephalopathy (PML), a rare, fatal, demyelinating disease. Individuals infected with HIV or taking prolonged immunomodulatory therapies have a heightened risk for developing PML. The cellular proteins and pathways utilized by JCPyV to mediate viral entry are poorly understood. Our findings further characterize how JCPyV utilizes the clathrin-mediated endocytosis pathway to invade host cells. We have identified specific components of this pathway that are necessary for the viral entry process and infection. Collectively, the conclusions increase our understanding of JCPyV infection and pathogenesis and may contribute to the future development of novel therapeutic strategies for PML.

Dataset Information

Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus.

Publications

Genetic and Functional Dissection of the Role of Individual 5-HT<sub>2</sub> Receptors as Entry Receptors for JC Polyomavirus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets