A second caveat which seriously affects the quality of the data is that the entity to be studied not be distorted by the ability of proteins to be manipulated and expressed in some form in E. coli. There are currently crystal structures of an E1-E2 fusion protein (PDB codes 3MUU), chikungunya glycoproteins (PDB codes: 3N40;3N41;3N42;3N43;3N44;2XFB;2XFC) and their fit into the SINV and/or Semliki Forest Virus cryo-reconstruction (PDB codes: 3MUW;2XFB;2XFC). These structures were produced by expressing the ectodomains of the E1 and E2 glycoproteins connected together by a linker. This fusion protein was constructed due to the inability of expressing E2 in E. coli. While it is common practice to remove troublesome domains from proteins to allow for crystallization to occur, how is it determined when there has been too much manipulation of the sequence? These structures have the same problems as E1*. The assumption that the transmembrane domains, which have been removed in the c DNA clone, and integration into the ER membrane play no role in the correct folding and assembly of the spike structure is an unproven assumption. For virus entry of the macromolecular SINV particle, structure is critical. The overall alphavirus icosahedral structure is unique in that it is a membrane containing virus that does not have the membrane exposed. The membrane itself is not the form determining factor as with influenza, and because it is beneath the outer protein shell is not readily available to fuse with host membranes. The essential role of the membrane may be to provide the scaffold upon which the virus is assembled. To fully explore the mechanism of virus penetration and entry other methods of analysis other than interpretations of structural fit can provide the missing events required for SINV infection.
Adsorption and receptor recognition
The process of virus attachment/absorption to the host cell is probably a multistep event. It is not a new proposal that virus infection begins with scanning of the cell surface that begins with a general “sticking” to the cell via one or more proteins (or the cell membrane itself) followed by “rolling” on the cell surface as it locates the proper receptor to initiate the penetration step of virus entry. This type of interaction has been identified recently using single particle fluorescence resonance energy transfer (FRET). SINV saturated with a membrane intercalated fluorescent self-quenching dye can be visualized when the dye is excited with a specific wavelength light. Fusion of virus with the cell membrane is detected when lipid mixing (fusion) releases the proteins and dequenching of the dye occurs causing it to fluoresce. Using this technique it has been shown that SINV moves on the cell surface in a neutral pH environment. If the pH of the medium bathing the cells is lowered the virus does not fuse with the cell surface as is the case with influenza, rather the virus “freezes” in place (K. Wenniger, unpublished observation). These observations indicate that viruses probe the cell surface for the proper receptor molecule.