PROPER SPACING BETWEEN HEPTAD REPEAT-B AND THE TRANSMEMBRANE DOMAIN BOUNDARY OF THE PARAMYXOVIRUS SV5 F-PROTEIN IS CRITICAL FOR BIOLOGICAL-ACTIVITY

The paramyxovirus, simian virus 5, fusion (F) protein contains seven a mino acids between heptad repeat a (a domain required for a biological ly active fusion protein) and the presumptive boundary of the transmem brane (TM) domain. The role of the seven membrane proximal residues in stability and fusion promotion was examined by construction of a seri es of insertion, substitution, and deletion mutants, as manipulation o f this region to enable proteolytic cleavage would facilitate producti on of a soluble F protein. The majority of the mutant F proteins both oligomerized and had kinetics of intracellular transport similar to th ose of wild-type (wt) F protein. All mutant F proteins were expressed at the cell surface at or near the same level as the wt F protein. How ever, by using both a qualitative lipid mixing assay and a quantitativ e content mixing assay for membrane fusion, it was found that mutant F proteins containing insertions in the region between heptad repeat a and the TM domain were unable to induce fusion, whereas the mutant F p roteins containing substitutions in this region, together with three o f the four mutants with deletions in this region, could induce fusion. Four of the F protein mutants contained a Factor Xa cleavage site, IE GR; however, Factor Xa treatment of cell surfaces released either none or only very small amounts (<1% of total protein) of the soluble hete rodimer F-1 + F-2. As an alternative method of generating soluble F pr otein, a glycosyl phosphatidylinositol (GPI) anchor was added to the F protein at three membrane-proximal positions. The highest level of su rface expression was observed when the final molecule did not contain a significant insertion of amino acids into the membrane proximal regi on. Two F-GPI mutants reached the surface at approximately 20% of the levels seen with the wt F protein, and approximately 25% of the cell s urface population of these mutants could be cleaved with phosphatidyli nositol phospholipase C (PI-PLC) to yield soluble F protein. However, all the F-GPI mutants oligomerized aberrantly and failed to promote fu sion. Taken together, these data indicate that the spacing of the regi on immediately adjacent to the presumptive boundary of the TM domain i s extremely important for the fusogenic activity of the SV5 F protein. (C) 1997 Academic Press.