MOLECULAR MECHANISM OF VIRUS NEUTRALIZATION

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The long-term objectives are to investigate the structure of virus glycoproteins and their involvement in attachment, penetration, and uncoating and to determine how they interact with antibody molecules to neutralize virus infectivity. Altough neutralization of virus has been studied emperically for many years, little is known about the molecular basis for this important natural host response to virus infection. Knowledge of this is a prerequisite to the understanding of how infectious virus-antibody complexes arise in vivo and how they contribute to the disease process of many viral infections. La Crosse, a medically important virus, has been chosen as a model to study the mechanism of antibody neutralization. It has two glycoproteins in its envelope, but only one of them, G1, is involved in antibody neutralization. G1 will be characterized by peptide mapping to determine sites of glycosylation, disulfide bonding, etc. Monoclonal antibodies have been prepared and used to map eight non-overlapping antigenic sites on G1. The exact location of these antigenic sites will be determined by using the monospecific antibodies to immunoprecipitate CNBr peptide fragments of G1, sequencing the amino terminus of each peptide, and correlating this with the deduced amino acid sequence of G1 as provided by others. It is proposed that there may be one main mechanism of neutralization but that the amount of virus neutralized and measured in plaque assays may be the sum of the probabilities that each virus-antibody complex will be inhibited from interacting with the cell at some level. To determine this, more must be known about the infectious entry of La Crosse virus. This will be done using binding penetration, and fusion assays in the presence or absence of lysosomotropic agents such as chloroquine. Virus reacted with each monoclonal antibody will be examined in these assays to determine where antibody must bind on G1 to neutralize virus and to determine at what step(s) in the multiplication cycle the virus replication is blocked. The possibility of neutralization at or just prior to transcription will also be examined. It is proposed that a conformational change in the structure of G1 is induced by antibody binding and that this is necessary for virus neutralization. Laser Raman spectroscopy will be used in attempts to determine if binding of antibody or synergistic binding of antibody pairs induces a conformational changes in G1 that is transmitted across the viral membrane.