Journal: Journal of Virology
Article Title: Role of Phosphatidylserine Receptors in Enveloped Virus Infection
Figure Lengend Snippet: Binding of lentiviral vectors mediated by soluble PtdSer-recognizing molecules. (A) Schematic representation of chimeric Sindbis virus Envs. The Sindbis virus Env is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into the viral envelope. Both the 2.2 and 2.2 1L1L pseudotypes are derived from the wild-type Sindbis virus Env with four mutations (red lines) to reduce their binding to original receptors while maintaining fusion activity. In addition, 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) at amino acid (a.a.) 70 of the E2 protein, and 2.2 contains the IgG-binding domain of protein A (ZZ), which was inserted into the E2 region at amino acid 70. (B) Expression levels of Axl (red line), integrin αVβ3 (blue line), and integrin αVβ5 (green line) were analyzed by flow cytometry after staining with antibodies specific to each antigen. The isotype of all antibodies was IgG1; thus, an IgG1 isotype control antibody was used for negative-control staining (black line). (C) Schematic representation of recombinant hGas6, hMFG-E8, mMFG-E8, and D89E, a mutant of MFG-E8 that cannot bind integrins. Gla, γ-carboxyglutamic acid domain; EGF, E1, and E2, epidermal growth factor-like domains; SHBG, sex hormone-binding globulin domain; C1 and C2, discoid domains; P/T, proline/threonine-enriched motif. The γ-carboxyglutamic acid domain of Gas6 and the C2 domain of MFG-E8 bind PtdSer. (D) Staining of HMVECs with an APC-conjugated anti-Flag tag antibody after incubation with hGas6 (red line), mMFG-E8 (blue line), or D89E (green line). The black line represents staining without prior incubation with any ligand. (E) Binding of the EGFP-labeled 2.2 1L1L pseudotype to HMVECs after incubation with hGas6, hMFG-E8, or mMFG-E8. Black line, HMVECs without virus; red line, HMVECs with virus with or without the bridging molecules indicated in the figure. The MFI values correspond to those for EGFP signals, which increase upon binding of EGFP-labeled virus. This experiment was repeated twice in singlicate and once in triplicate, and the MFI values shown are the averages and standard deviations of the triplicate experiment; representative flow cytometry histograms are shown. Significance was calculated by comparing the value of virus binding without a bridging molecule to the value of virus binding in the presence of the bridging molecules indicated in the figure using a two-sample Student t test (**, P
Article Snippet: Enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors pseudotyped with various Envs were produced by transfecting 293T cells with an Env expression vector (Sindbis, 2.2, 2.2 1L1L, VSV envelope G protein [VSV-G], gp64, or RRV), the packaging plasmid PAX2 (Addgene), and a lentiviral vector (cppt2e) containing EGFP as its transgene ( ).
Techniques: Binding Assay, Synthesized, Derivative Assay, Activity Assay, Expressing, Flow Cytometry, Cytometry, Staining, Negative Control, Recombinant, Mutagenesis, FLAG-tag, Incubation, Labeling