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Figure 4 | Retrovirology

Figure 4

From: Single amino acid substitution (G42E) in the receptor binding domain of mouse mammary tumour virus envelope protein facilitates infection of non-murine cells in a transferrin receptor 1-independent manner

Figure 4

Mutation of a single amino acid in the RBS alters the tropism of egfp-containing MMTV-based vector. (A) MMTV Env carrying G42E mutation is expressed and cleaved into SU (gp52) and TM (gp36) subunits. Equivalent protein content (10 μg) of whole cell extracts from wild-type and mutant pENVC3H-transfected HEK293T cells were subjected to SDS-PAGE followed by Western blotting analysis with anti-gp52/36 (1:12000). (B) Target cells (Hs578T or NMuMG) were transduced with MMTV-based vector particles carrying wild-type or mutant MMTV(C3H) envelope. Transduction levels were made relative to the value obtained for MMTV-based vector particles carrying wild-type Env and expressed as a percentage of that control. The values shown are mean ± standard errors from three independent experiments. The inset shows a Western blot of 10 μl of each concentrated virus preparation probed with polyclonal anti-MMTV-CA antiserum (1:4000). (C) Hs578T cells were transduced with MMTV vectors carrying wild-type MMTV(C3H) Env, mutant MMTV(C3H) Env or MLV 4070A Env. Neutralization, heat inactivation and AZT treatment were performed as described in materials and methods. Transduction levels were made relative to the value obtained for the infected cells and expressed as percentage of that control. The values shown are mean ± standard errors from three independent experiments. (D) MMTV-based vector particles carrying either wild-type or mutant MMTV(C3H) Env were incubated with the indicated amount of heparan sulfate, and mixture was subsequently used to transduce Hs578T cells. Transduction levels were made relative to the value obtained in the absence of heparan sulfate and expressed as a percentage of that control. The values shown are mean ± standard errors from three independent experiments. Transduction was detected by flow cytometry (FCM) 3 days post infection. The number of eGFP expressing cells was counted using forward scatter vs. fluorescence intensity in the FL1 channel (eGFP) plots.

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