Single and multiple deletions in the transmembrane domain of the Sindbis virus E2 glycoprotein identify a region critical for normal virus growth

AbstractSindbis virus is composed of two nested T = 4 icosahedral protein shells containing 240 copies each of three structural proteins: E1, E2, and Capsid in a 1:1:1 stoichiometric ratio. E2 is a 423 amino acid glycoprotein with a membrane spanning domain 26 amino acids in length and a 33 amino acid cytoplasmic endodomain. The interaction of the endodomain with the nucleocapsid is an essential step in virus maturation and directs the formation of the outer protein shell as envelopment occurs. A previous study had determined that deletions in the transmembrane domain could affect virus assembly and infectivity (Hernandez et al., 2003. J. Virol. 77 (23), 12710–12719). Unexpectedly, a single deletion mutant (from 26 to 25 amino acids) resulted in a 1000-fold decrease in infectious virus production while another deletion of eight amino acids had no affect on infectious virus production. To further investigate the importance of these mutants, other single deletion mutants and another eight amino acid deletion mutant were constructed. We found that deletions located closer to the cytoplasmic (inner leaflet) of the membrane bilayer had a more detrimental effect on virus assembly and infectivity than those located closer to the luminal (outer leaflet) of the membrane bilayer. We also found that selective pressure can restore single amino acid deletions in the transmembrane domain but not necessarily to the wild type sequence. The partial restoration of an eight amino acid deletion (from 18 to 22 amino acids) also partially restored infectious virus production. The amount of infectious virus produced by this revertant was equivalent to that produced for the four amino acid deletion produced by site directed mutagenesis. These results suggest that the position of the deletion and the length of the C terminal region of the E2 transmembrane domain is vital for normal virus production. Deletion mutants resulting in decreased infectivity produce particles that appear to be processed and transported correctly suggesting a role involved in virus entry