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Figure 2 | Retrovirology

Figure 2

From: Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses

Figure 2

Over-expression of REAF confers restriction which is viral route of entry dependent. (A) Over-expression of REAF restricts early HIV-1 DNA production. HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-GFP antibody. Transfection of REAF-EGFP results in expression of REAF (110, 160 and 250 kDa including EGFP tag (30 kDa). Endogenous REAF was knocked out of HeLa-CD4 cells using siRNA targeting its 3’ UTR. (B) Infected cells were measured following transient over-expression of REAF-EGFP or pEGFP-C3 and challenge with HIV-189.6. Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate. (C) Transient over-expression of REAF-EGFP in HeLa-CD4 cells results in decreased RU5 and late RT products 8 hr post challenge with HIV-189.6. HIV-1 DNA copies are normalised to genomic GAPDH and presented per million cells. Results are mean ± s.d. of a representative experiment performed in duplicate. (D) Restriction by REAF is dependent upon viral route of entry. HIV-189.6Δenv was pseudotyped with VSV-G envelope and used to challenge HeLa-CD4 cells following REAF siRNA knockdown. p24 immunostaining was used to detect FFU compared with HIV-189.6. Results are shown as FFU/ml and are mean ± s.d. of a representative experiment performed in duplicate.

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