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. 2015 Nov 25;90(3):1647-56.
doi: 10.1128/JVI.02013-15. Print 2016 Feb 1.

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

Kristina Pagh Friis  1 , Xavier Iturrioz  2 , Jonas Thomsen  3 , Rodrigo Alvear-Perez  2 , Shervin Bahrami  4 , Catherine Llorens-Cortes  2 , Finn Skou Pedersen  5
Affiliations

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

Kristina Pagh Friis et al. J Virol. .

Abstract

We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization.

Importance: Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry.

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Figures

FIG 1
FIG 1
Molecular evolution of SL3-AP. (A) Schematic showing the location and amino acid sequence of the apelin cassette in the SL3-2 Env protein (16). VRA, VRB, and VRC are variable regions A, B, and C, respectively. The apelin sequence is green, and the linker sequence is blue; the black sequences are of SL3-2 origin. LTR, long terminal repeat. (B) Illustration of the strategy used for molecular evolution studies with SL3-AP. (C) Infectivity after passaging of SL3-AP on cells expressing one of the two receptors, i.e., Xpr1 on NIH 3T3 cells or hAPJ on D17 hAPJ cells. The infectivity of the passaged virus Xpr1 N10 and hAPJ N10 was measured on NIH 3T3, D17 hAPJ, and D17 cells by flow cytometry with an antibody against the MLV Env protein. N10 indicates that the virus has been through 10 passages in the respective cell line. The data are representative of three independent experiments. PE, phycoerythrin.
FIG 2
FIG 2
Selected amino acid changes found in hAPJ molecular evolution studies. (A) Alignment of genomic DNA sequences from 10 passages of SL3-AP in D17 APJ cells reveal recurrent point mutations. The selected mutations are shown on the arrows in the passages (P1 to P6) where they became prevalent and not in the passages where they first arose. (B) The positions of the mutations in the SL3-2 AP Env protein are illustrated on the base pair level, as well as in the amino acid sequence. Black, SL3-2 Env; blue, SGGSG linker sequence; green, apelin-13 ligand. The names refer to the positions in the amino acid sequence of SL3-2 AP Env, as illustrated.
FIG 3
FIG 3
Infectivity of the SL3-2 AP mutants expressed in the minivirus system on noninfected NIH 3T3 or D17 hAPJ cells measured as neomycin resistance (neoR). No significant variation is registered on NIH 3T3 or D17 hAPJ cells. wt, wild type.
FIG 4
FIG 4
Surface expression of Env on D17 hAPJ cells infected with virions expressing the chimeric Env proteins and pseudotyped with VSV-G. Data from two independent experiments out of four are shown.
FIG 5
FIG 5
125I-pE13F saturation binding curve for a D17 hAPJ membrane preparation. The saturation experiment was conducted with D17 hAPJ membrane preparations and increasing concentrations of 125I-pE13F with or without 10−6 M K17F to define specific binding. Kd and Bmax were deduced from the specific binding curve.
FIG 6
FIG 6
Internalization of rat apelin receptor-EGFP by apelin peptides G14F and G14L. CHO cells stably expressing rat apelin receptor-EGFP were stimulated with different concentrations of apelin peptides G14F and G14L as indicated. The cells were fixed and analyzed by confocal microscopy. The results are representative of several independent experiments.
FIG 7
FIG 7
Internalization of human apelin receptor-EGFP by apelin peptides G14F and G14L. D17 cells stably expressing human apelin receptor-EGFP were stimulated with different concentrations of apelin peptides G14F and G14L as indicated. The cells were fixed and analyzed by confocal microscopy. The results are representative of five independent experiments.
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