W0295

Visualization of Membrane Protein Domains in Enveloped Viruses. Michael G. Rossmann¹, Suchetana Mukhopadhyay¹, Wei Zhang¹, Paul R. Chipman¹, Ying Zhang¹, Jeroen Corver², Peter R. Johnson¹, Timothy S. Baker¹, James H. Strauss², Richard J. Kuhn¹, ¹Dept. of Biological Sciences, Purdue Univ., West Lafayette, IN 47907-2054 and ²Div. of Biology, California Inst. of Technology, Pasadena, CA 91125.

There are only a few examples of membrane proteins whose structures have been determined in situ. However, an increasing number of membrane structures have been determined in two- or three-dimensional hydrophobic environments. Improved technology for reconstructing cryo-electron microscopy images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. We will show the structures of Sindbis virus (an alphavirus) and dengue and yellow fever viruses (flaviviruses) whose structures we have determined to about 9 Å resolution, enabling us to fit the various known atomic resolution membrane protein structures into the lipid envelope of these viruses.

Flaviviruses have two transmembrane proteins. The E (envelope) protein includes the fusion peptide and a receptor recognition domain. The immature prM (pre-membrane) protein is cleaved in the maturation step, making the newly assembled particle infectious and leaving only the M protein in the viral capsid. The α-helical “stem” regions of E and M were found buried in the outer leaflet of the viral membrane. The “anchor” regions of E and M proteins each form antiparallel EE and MM transmembrane α-helices leaving their carboxy termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein.

The well-resolved E1-E2 transmembrane regions of Sindbis virus form α-helical coiled coils that are consistent with the T = 4 symmetry of the virus. In alphaviruses, in contrast to flaviviruses, the carboxy end of E2 has been withdrawn to the cytoplasmic side of the membrane where it associates with the nucleocapsid. Furthermore, in alphaviruses the E1 and E2 proteins associate with each other to form parallel coiled coils, whereas in flaviviruses the E and M proteins remain apart from each other within the viral membrane.