Preliminary Analysis of Vital Proteins of the Human Respiratory Syncytial Virus

Abstract

Human Respiratory Syncytial Virus infects the vast majority of children under the age of two and reoccurs in adulthood. It is a serious global problem as severe infection and death can result in the very young or very old and in immunocompromised people. The related bovine Respiratory Syncytial Virus is a serious problem for the farming community. To date, there is no effective treatment for Respiratory Syncytial Virus infection, with drugs only available to reduce symptoms in the most severe cases. Human Respiratory Syncytial Virus produces 10 proteins carrying out a diverse array of roles for the infectivity of the virus. Despite this there is still a poor understanding of the three dimensional structure. Only two proteins have been resolved to atomic resolution, the Nucleocapsid protein and the Matrix protein. The objective of this thesis is to initiate work on a further two proteins, with the long term aim of crystallisation studies that may lead to high resolution structures. The Fusion transmembrane glycoprotein is responsible for the fusion of the virion membrane to the host cell membrane for viral entry and for the fusion of an infected cell membrane to membranes of healthy cells, spreading infectivity. Without the Fusion protein there is no infectivity and it is therefore a therapeutic target. Structural information such as a high resolution X-ray crystal structure is required to facilitate the design of inhibitors that could be future therapeutic agents. The second protein studied here is the M2-1 protein, which is a transcriptional elongation factor, involved in replication of the viral genome. Deletion of M2-1 has adverse affects on the replication of the virus. Therefore M2-1 could also make a strong target for drug design. This thesis explores what is currently known about each of these vital proteins, the advantages in inhibiting their respective activities and the possible routes to effective inhibitor design. Procedures for expression and purification of the M2-1 protein are detailed here, as are preliminary experiments into the cloning and expression of the Fusion protein in insect cells. Immunofluorescence experiments into the localisation of the Fusion protein within mammalian cells and the possible interaction of the Fusion protein with the Matrix protein are thoroughly described.