Short-Stalk Isoforms of CADM1 and CADM2 Trigger Neuropathogenic Measles Virus-Mediated Membrane Fusion by Interacting with the Viral Hemagglutinin

Abstract

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae , usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We have recently shown that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors. Importance Measles, an acute febrile illness with skin rash, is still an important cause of childhood morbidity and mortality worldwide. Measles virus (MeV), the causative agent of measles, may also cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. The disease is fatal, and no effective therapy is available. Recently, we have reported that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV cell-to-cell spread in neurons. These molecules interact in cis with the MeV attachment protein on the same cell membrane, triggering the fusion protein and causing membrane fusion. CADM1 and CADM2 are known to exist in multiple splice isoforms. In this study, we report that their short-stalk isoforms can induce membrane fusion by interacting in cis with the viral attachment protein independently of its receptor-binding head domain. This finding may have important implications for cis -acting fusion triggering by host factors.

Collective fusion activity determines neurotropism of an en bloc transmitted enveloped virus

“…Following procedures previously described in ( 14 ), 293FT cells cultured on 12-well plates were transfected with 0.5 μg of pCA7 encoding one of FLAG-tagged F proteins with 0.5 μg of pCA7 encoding E-cadherin (a cell surface protein control) using Lipofectamine LTX. At 24 hours after transfection, cells were washed with phosphate-buffered saline (PBS) and then incubated with 200 μl of the biotin reagent solution [2 mM EZ-Link N -hydroxysulfosuccinimide (sulfo-NHS)–biotin (Thermo Fisher Scientific) in PBS] for 30 min at 4°C.…”

“…Following procedures previously described in ( 14 ), proteins in samples were separated by SDS–polyacrylamide gel electrophoresis and then blotted onto polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). The membranes were incubated with primary antibodies (Abs) for 1 hour.…”

“…The H protein binds to receptors on the human cells and subsequently triggers the conformational changes of the trimeric F protein from the prefusion form to the postfusion form, leading to virus-to-cell or cell-to-cell membrane fusion. While the wild-type (WT) F protein cannot induce membrane fusion in neurons lacking MeV receptors [signaling lymphocytic activation molecule family member 1 (SLAMF1) and nectin-4], hyperfusogenic mutant F proteins from SSPE patientderived MeV strains can do so by using cell adhesion molecule 1 (CADM1) and CADM2 as "cis-acting receptors" (host factors triggering fusion in cis) (12)(13)(14). The H protein interacts with CADM1 and CADM2 in cis on the same membrane, triggering the conformational changes of the hyperfusogenic mutant F proteins but not of the WT F protein.…”

Novel and classical morbilliviruses: Current knowledge of three divergent morbillivirus groups

“…In MeV infection, there is a late complication called subacute sclerosing encephalitis (SSPE) that arises from persistent infection in the brain, and viruses isolated from patients with SSPE have mutant fusion (F) proteins with high fusion ability. These mutant F proteins with high cell fusion ability are expressed on the same cells as cell adhesion molecule (CADM) 1 and 2 (called “cis” interactions), leading to cell fusion through the binding actions of these molecules 122,123 . Because these mutant F proteins differ in ability from the field strains, 124 it remains unclear whether natural morbillivirus infection in the CNS utilizes a similar mechanism.…”

“…These mutant F proteins with high cell fusion ability are expressed on the same cells as cell adhesion molecule (CADM) 1 and 2 (called "cis" interactions), leading to cell fusion through the binding actions of these molecules. 122,123 Because these mutant F proteins differ in ability from the field strains, 124 it remains unclear whether natural morbillivirus infection in the CNS utilizes a similar mechanism. However, experiments using F proteins isolated from patients with measles inclusion body encephalitis (F proteins containing a mutation for high cell fusion capacity) showed that MeV strains with these mutated F proteins caused CNS invasion through the respiratory route, 125 suggesting that the cell fusion capacity of the F protein is related to its neuropathogenic potential.…”