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The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins

Byoung Y. Ryu

Tatiana Zavorotinskaya

Bernadette Trentin

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, USA

Correspondence
Lorraine M. Albritton
lalbritton{at}utmem.edu

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

Present address: Department of Hematology, St Jude Children's Research Hospital, Memphis, TN, USA.

Present address: Chiron Corporation, Emeryville, CA, USA.

Present address: CNRS UMR5121 UM1, Institut de Biologie, 4 boulevard Henri IV, 34000 Montpellier, France.

Supplementary figures are available with the online version of this paper.

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