Production and analysis of alphaviral glycoprotein-pseudotyped retroviruses

Alphaviruses, are arthropod-vectored pathogens, some of which pose a significant health risk to humans. Alphaviruses have a lipid envelope in which two glycoproteins are anchored. The E2 glycoprotein binds to the surface of a susceptible cell, while the E1 glycoprotein catalyzes the fusion of the viral membrane with the cellular membrane. Thereby, the internal components of the alphavirus are delivered to the cytoplasm of a host cell. Moloney-Murine Leukemia virus is a retrovirus. Retroviruses are also enveloped by a lipid membrane and possess glycoproteins that bind to the surface of susceptible cells and catalyze the fusion of viral and cellular membranes. We have constructed chimeric viruses containing the internal components from Moloney-Murine Leukemia virus and enveloped by a membrane in which the alphaviral E2 and E1 glycoproteins are anchored. Alphaviral glycoproteins mediate the binding of these chimeras to the surface of cells, as well as the delivery of the internal Moloney-Murine Leukemia virus capsid and genome. The chimeric viruses have an expanded range of cells that are susceptible to their infection. We have determined that alphaviral glycoprotein-pseudotyped retroviruses are useful as tools for the delivery of genes to cells in-vitro and in-vivo. We also used the pseudotyped viruses to investigate the activity of both alphaviral envelope glycoproteins E2 and E1. We have determined that these alphaviruses infect cells through the endosomal uptake pathway. We used Ross River virus glycoprotein-pseudotyped Moloney-Murine Leukemia virus to investigate the sequence requirements of the membrane-spanning domain from the E1 glycoprotein for the function of the glycoprotein. The amino-acid sequence of this membrane-spanning domain is important for the catalysis of membrane fusion by Ross River virus envelope proteins. The sequence requirements that we have identified agree with those determined for the Moloney-Murine Leukemia virus fusion protein. We propose a model in which both a bend in the membrane-spanning domain, and the recruitment of H2O into the intraleaflet space are fundamental to the catalysis of membrane fusion by a variety of unrelated proteins