JC Polyomavirus Entry by Clathrin-Mediated Endocytosis Is Driven by β-Arrestin

Abstract

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 ␣2,6-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-HT 2 Rs), 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-HT 2 R endocytic machinery, including ␤-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 ␤-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a ␤-arrestin binding domain (Ala-Ser-Lys) within the intracellular C terminus of 5-HT 2A R severely diminished internalization and infection, suggesting that ␤-arrestin interactions with 5-HT 2A R are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrinmediated 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 clathrinmediated 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.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

“…The ubiquitously expressed ␤-arrestin1 and ␤-arrestin2 are best known for functioning in the regulation of phosphorylated GPCR desensitization and internalization (52). To date, ␤-arrestin1 and ␤-arrestin2 have been reported to be involved in the internalization process of many viruses, such as HIV (55) and JC polyomavirus (56). The GPCR family proteins have different binding affinities for ␤-arrestin1 and ␤-arrestin2 (53).…”

Serotonergic drugs inhibit CHIKV infection at different stages of the cell entry pathway

“…This has been linked to alterations in the protein kinase A (PKA) pathway which interfered with claudin 1, an important receptor for post-binding steps of hepatitis C virus cell entry (17,49). For JC polyomavirus, 5-HT2 receptor antagonists inhibited infection due to interference of binding of β-arrestin to the 5-HT2A receptors, which is required for internalization of the virus via clathrin-coated vesicles (20,51,52). During reovirus infection, 5-NT strongly inhibited the cell entry of reovirus whereas MM enhanced reovirus infectivity.…”

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

“…JCPyV does not seem to interact with 5-HT 2 Rs at the plasma membrane because JCPyV binding to cells overexpressing the 5-HT 2 Rs is not enhanced ( Assetta et al, 2013 ). JCPyV enters host cells via clathrin-mediated endocytosis, and the 5-HT 2 Rs are also internalized by the same mechanism ( Mayberry et al, 2019 ; Pho et al, 2000 ; Querbes et al, 2004 ). It is not yet known whether there is an interaction between JCPyV and the 5-HT 2 Rs during entry, and studies to clarify whether there is a redundant role for each individual isoform in the context of JCPyV infection of glial cells have not been performed.…”

“…It is not yet known whether there is an interaction between JCPyV and the 5-HT 2 Rs during entry, and studies to clarify whether there is a redundant role for each individual isoform in the context of JCPyV infection of glial cells have not been performed. Additionally, it is not known what structural domains of the 5-HT 2 Rs are crucial for JCPyV infection, although recently a motif in the C terminus of the 5HT 2A receptor was shown to be important for virus internalization and infection ( Mayberry et al, 2019 ). In this study, mutagenesis of an ASK (Ala-Ser-Lys) motif in the C-terminal tail of 5HT 2A R and small interfering RNA(siRNA) knockdown of beta-arrestin reduced JCPyV infection ( Mayberry et al, 2019 ).…”