Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal uid (CSF) of MS patients contribute to in ammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-a nity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identi ed by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSFderived antibodies against MS-associated viruses. Sequence analysis, a nity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM crossreactive antibody. Molecular mimicry is facilitated by a post-translational modi cation of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies. Main TextThe presence of oligoclonal bands (OCB) in cerebrospinal uid (CSF) and the e cacy of B cell depleting therapies emphasize the importance of B cells in the pathobiology of multiple sclerosis (MS) 2 . Anti-viral antibodies against mumps, measles, varicella-zoster, and Epstein-Barr Virus (EBV) are often present in MS 4,5 , but their relevance is unclear. Anti-EBV antibody titers in over 99% of MS patients provide evidence for an epidemiological link between MS and EBV 6 . Symptomatic infectious mononucleosis during EBV infection increases risk for MS 7 . Molecular mimicry between virus and self-antigens is a potential mechanism that might explain this association 8 . Antibodies against certain EBV nuclear antigen 1 (EBNA1) regions have been found in MS patients, including the region AA365-426 5,9-12 , which we describe here in our identi cation of molecular mimicry between EBNA1 and the glial cellular adhesion molecule GlialCAM. The potential signi cance of this mimicry in the pathophysiology of MS is described in detail.The B cell repertoire in MS CSF plasmablasts is highly clonal CSF and blood samples were obtained from MS patients during the onset of disease (clinically isolated syndrome, n=5) or an acute episode of relapsing-remitting MS (n=4). Patients with a CSF pleocytosis of >10 cells/µl were selected (Extended Data Table 1, Supplementary Discussion). Single B cells were sorted by ow cytometry (Extended Data Fig. 1a,b). Characteristic phenotypic differences of B cells in blood and CSF were observed 13,14 , including (i) high plasmablast (PB) counts in CS...

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.

T he replication cycle of influenza A virus (IAV) is complex. The virus attaches to susceptible host cells via its hemagglutinin (HA), a homotrimeric type I membrane glycoprotein that recognizes sialoconjugates (1-3). The virus then enters the endocytic pathway, and upon arrival in acidified late endosomes, the HA trimer undergoes a conformational transition that renders it fusogenic. The M2 ion channel is responsible for acidification of the virus lumen, which results in dissociation of the eight viral ribonucleoproteins (vRNPs) (comprised of PB1, PB2, PA, NP, and genomic RNA) from the M1 protein and release of the vRNPs into the host cytosol (4-6). These vRNPs translocate into the nucleus via one of at least two nuclear localization sequences, NLS1 and NLS2, in NP (7-11). mRNA generated from vRNP-dependent synthesis of viral genomic RNA (vRNA) is exported from the nucleus and translated in the cytoplasm. Newly synthesized PB1, PB2, PA, and NP translocate into the nucleus as monomers (NP and PB2) or dimers (PB1-PA), where they assemble with newly synthesized vRNA to yield the vRNP complex (12, 13). These vRNP complexes are exported from the nucleus for incorporation into budding virus particles (14).In the course of a single replication cycle, influenza virus NP interacts with viral RNA and with viral proteins, including PB1, PB2, and M1 (15, 16). Several host proteins also interact with NP, including importin-␣, BAT1,. Mapping such interactions and assessing their relevance for virus replication remains a challenge because of their often-essential role in the replication cycle. With rare exceptions, the influenza virus genome has resisted genetic manipulation, because many such changes cause a complete loss of a particular function (21-23) and compromise viral fitness.The variable domains of heavy-chain-only antibodies (VHHs) isolated from camelids are small, ϳ15 kDa, and their ability to bind their cognate ligand is largely independent of modifications such as disulfide bonds and glycosylation (24,25). These properties allow the VHHs to be expressed in the cytosol of eukaryotic cells with retention of the antigen binding capabilities. This in turn permits the specific targeting of host or viral proteins recognized by VHHs, thus enabling possible perturbation of target protein function (26-32; for a review, see reference 33). VHHs are