SARS coronavirus membrane proteins

Abstract

Membrane proteins play an essential role in the coronavirus life cycle. The scope of this thesis was to obtain further insight into membrane-associated processes during SARS-CoV infection by studying various features of different viral membrane proteins. Targets were selected mainly from two different classes of membrane-associated proteins, i.e. replicase (nonstructural) proteins and accessory (or group-specific) proteins. The three hydrophobic nonstructural proteins (nsp) were characterized in order to obtain insight into the membrane anchoring of the coronavirus replication complex. These proteins, nsp3, nsp4 and nsp6, are predicted to represent key components directing the formation of the viral replication structures. Therefore the biosynthesis, processing and membrane topology of each one of these proteins was studied. The analysis of nsp4 additionally focused on the localization of this protein to replication sites and on the contribution of the host cell to the formation of these sites. The membranes of the replication structures appeared to be derived from the endoplasmic reticulum and the generation of these structures was found to involve the cellular early secretory pathway. The study of nsp3 and nsp6 concentrated particularly on the membrane integration of these complex proteins and on the contribution of each of the hydrophobic domains to this process. Both proteins appeared to contain a hydrophobic domain that fulfills the criteria for a transmembrane domain but which does not actually span the lipid bilayer. This is more often found in RNA virus nonstructural proteins and might be related to functions of these proteins. These domains could be involved in protein-protein interactions or in the shaping of the membrane. Also the SARS-CoV accessory proteins were studied. The accessory protein encoded by ORF3a, which had just been shown to be incorporated into virions, was characterized. In view of its remarkable structural similarity to the membrane protein M, we comparatively analyzed the co- and posttranslational modifications of these two proteins. Both proteins were found to be glycosylated although differentially. Whereas the SARS-CoV M protein was N-glycosylated, the 3a protein was modified by O-linked sugars. Remarkably, in this aspect the 3a protein resembles the M proteins of the most closely related coronaviruses more than the SARS-CoV M protein does. Finally, the functional expression of the ORF8 related protein products was examined. This genomic region is of particular interest in the evolution of the SARS-CoV, since a deletion occurred early during the SARS epidemic, which was found in all later human virus isolates. Because of this deletion only a very small fragment of the original protein, 8ab+, is still produced. The 8ab+ protein found in animal viruses accumulates in the ER, where it is N-glycosylated and forms homomultimeric complexes. The deletion clearly had a radical effect on the functional meaning of this genomic region. However, whether and how this influenced the spread of the virus within the human population remains to be determined