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European Heart Journal 1991 12(Supplement D):60-64; doi:10.1093/eurheartj/12.suppl_D.60
Copyright © 1991 by the European Society of Cardiology.
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© 1991 The European Society of Cardiology

Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to coxsackievirus B4

K. W. Beisel*, J. Srinivasappa{dagger},{ddagger} and B. S. Prabhakar{dagger},

* Department of Pathology and Microbiology, University of Nebraska Medical Center Omaha, Nebraska
{dagger} Laboratory of Oral Medicine, National Institute of Dental Research, National Institutes of Health Bethesda, Maryland, U.S.A.

Address for correspondence: B.S. Prabhakar, Dept. of Microbiology, Microbiology Building, UTMB, Galveston, TX-77550, U.S.A.

A panel of coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies was tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. Immunohistochemical studies revealed an A band pattern of staining of the heart by this antibody. Western immunoblotting of sequential differential extracts of heart showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin.

The monoclonal antibody 356-1 was then used to screen a Agt11 CD1 mouse heart cDNA expression library. Forty-eight positive plaques were obtained, 14 of which reacted strongly with the antibody and were selected for additional studies. In 13/14 clones, inserts were amplified using the polymerase chain reaction (PCR). The PCR products ranged in size from ~150 to 1400 bp. Northern hybridization using these inserts demonstrated that 10/13 recognized a ~6·5 kb message in mouse heart total RNA and not in liver total RNA. These amplified inserts were sequenced and were found to contain sequences which encode ~1/3 of the amino-terminal end of light meromyosin. By comparison with known sequences of rat alpha and beta cardiac myosin heavy chains, we were able to identify sequences specific for alpha chain as potential cross-reactive autoantigenic epitope. This putative antigenic site is located between amino-acid residues 1304 and 1647 of the alpha cardiac myosin encoded by the cDNAs. These studies imply that molecular mimicry is one mechanism by which autoimmunity might develop during CVB4 myocarditis.

Key Words: Alpha cardiac myosin heavy chain • autoantigen • coxsackievirus B4 • light meromyosin • {lambda}gt11 • monoclonal antibody • recombinant protein

{ddagger} Present address: Oxford Veterinary Laboratories, Inc., Number One Bio Drive, P.O. Box 775, Worthington, MN 56187, U.S.A.


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