Progressive Multifocal Leukoencephalopathy-Associated Mutations in the JC Polyomavirus Capsid Disrupt Lactoseries Tetrasaccharide c Binding

Abstract

The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the α2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious.

Structure Analysis of the Major Capsid Proteins of Human Polyomaviruses 6 and 7 Reveals an Obstructed Sialic Acid Binding Site

“…These findings are consistent with the crystal structure analysis, and they are also in agreement with experiments employing crystal soaking and glycan array screening, neither of which (30,51). (C and F) JCPyV L54F is a VP1 mutant with an abolished sialic acid binding site (37) and was used to test for sialic acid-independent cell binding. FITC, fluorescein isothiocyanate; max, maximum.…”

“…4C and F). This mutant has a disrupted VP1 sialic acid binding site and no longer engages sialylated receptors (37).…”

“…Four nonnative residues (GSHM) are present at the N termini of both proteins after purification, and a nonnative glutamine forms the C terminus of HPyV7 VP1. For cell binding experiments, the JC polyomavirus (JCPyV) VP1 wild type and L54F mutant (residues 22 to 289) and murine polyomavirus VP1 (RA strain; residues 33 to 316) were expressed and purified accordingly (30,37).…”

Interferon-Regulatory Factor 5-Dependent Signaling Restricts Orthobunyavirus Dissemination to the Central Nervous System

“…8F and G). The persistence of OROV in different peripheral organs in Irf5 Ϫ/Ϫ mice could lead to the selection of an encephalitic variant of OROV with greater neuroinvasive potential, as seen with the JC polyomavirus (32,33). To test this idea, we homogenized brain tissue from OROV-infected Irf5 Ϫ/Ϫ mice and used this to infect WT and Irf5 Ϫ/Ϫ mice.…”

An animal model for progressive multifocal leukoencephalopathy

“…Moreover, infection of the chimeric mice with JCV mutants derived from human patients also led to viral spread and disease (30,31). This latter point is interesting, as mutant virus arising in human patients seems unable to recognize sialic acid-containing receptors for the virus (32,33). These viruses must evolve to use alternative means of infection and spread, the mechanisms of which are under investigation in several labs.…”