Prediction and Identification of a Permissive Epitope Insertion Site in the Vesicular Stomatitis Virus Glycoprotein

Abstract

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertiontolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.

Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

“…Next, to isolate insertion mutants that presented functionally active peptides, we repeated the selection protocol but also purified the viral library on a Ni-NTA column before infecting cells. This procedure was repeated until the majority of the viral library To assess the ability of VSV-G-His 6 variants to pseudotype retroviral and lentiviral vectors, we inserted a His 6 tag into all three novel sites that were identified from the selection as well as in site 17, the N terminus of the mature protein, and in two previously identified sites, the temperature-sensitive site 25 (14) and site 191 (37). Furthermore, to explore the effects of different amino acid linkers flanking the His 6 tag, we constructed multiple variants at two of the sites (Table 1).…”

“…Novel functions have been engineered into various envelope proteins through the introduction of new sequences at known binding domains or known tolerated insertion sites (7,45). Modifications to the extracellular domain of VSV-G, however, have been hindered by a lack of structural knowledge of the protein and limited identification of permissible insertion sites (14,28,37), making it an excellent candidate for applying a novel library mutagenesis method to improve its functionality.…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…However, with few exceptions (Hatziioannou et al, 1999;Patterson et al, 1999;Russell and Cosset, 1999), an incomplete understanding of the complicated structurefunction relationships of viral envelopes still limits our ability to rationally engineer targeted and efficient envelope proteins. For example, the binding of the envelope protein to a cell surface receptor often triggers viral fusion with the cell membrane, and the complex fusion domains can be widely distributed throughout an envelope protein (Kayman et al, 1999;Li et al, 1993;Martinez and Wertz, 2005;Schlehuber and Rose, 2004). Swapping binding specificity without compromising the actuation of fusion is challenging.…”

Rapid Adaptation of a Recombinant Vesicular Stomatitis Virus to a Targeted Cell Line

“…Twenty-four hours later, the medium was removed from each well and replaced with 1 ml of cysteine/methionine-free media (Cellgro catalog no. 17-104-L1; Mediatech, Inc., Herndon, VA) containing 20 Ci of [ 35 S]methionine (10 mCi/ml, Trans 35 S-LABEL, catalog no. 510067; MP Biomedicals, LLC, Irvine, CA).…”

“…VSV pseudotypes were created by transfecting BHK-21 with DNA encoding either the initial gp or the adapted gp and then infecting them with rrVSV whose genome did not contain any gp gene. Preparation of 35 S-labeled pseudotype virus was performed as previously described (3). Briefly, BHK-21 cells were transfected with vectors expressing one of the chimeric Sindbis gp using a standard Lipofectamine protocol and incubated at 32°C.…”