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Journal of Virology, February 2000, p. 1686-1693, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Central Proline of an Internal Viral Fusion Peptide Serves Two Important Roles

S. E. Delos,1 J. M. Gilbert,2 and J. M. White1,*

Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908,1 and Department of Pathology, Harvard Medical School, Boston, Massachusetts 021152

Received 13 July 1999/Accepted 19 November 1999

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta -sheet), a tight reverse turn containing the proline, and a second region of order (alpha -helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.

* Corresponding author. Mailing address: Department of Cell Biology, University of Virginia Health System, School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732. Phone: (804) 924-2593 or (804) 924-2009. Fax: (804) 982-3912. E-mail: jw7g{at}unix.mail.virginia.edu.

Journal of Virology, February 2000, p. 1686-1693, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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