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. 2010 Sep;84(17):8980-5.
doi: 10.1128/JVI.00858-10. Epub 2010 Jun 16.

Potent Lentiviral Restriction by a Synthetic Feline TRIM5 Cyclophilin A Fusion

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Free PMC article

Potent Lentiviral Restriction by a Synthetic Feline TRIM5 Cyclophilin A Fusion

Isabelle Dietrich et al. J Virol. .
Free PMC article

Abstract

A synthetic feline TRIM5-cyclophilin A fusion protein (feTRIMCyp) was generated and transduced into feline cells. feTRIMCyp was highly efficient at preventing infection with human (HIV) and feline (FIV) immunodeficiency virus pseudotypes, and feTRIMCyp-expressing cells resisted productive infection with either FIV-Fca or FIV-Pco. The restriction of FIV infection by feTRIMCyp was reversed by the cyclosporine (Cs) derivatives NIM811 and Debio-025 but less so by Cs itself. FeTRIMCyp and FIV infections of the cat offer a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.

Figures

FIG. 1.
FIG. 1.
Restriction of lentiviral entry by feline TRIMCyp and its reversal by CypA antagonists. CrFK (ID10) cells were stably transduced with a retroviral vector bearing feline TRIMCyp (square) or vector only (circle). The cells were challenged with serial dilutions of HIV(VSV) (A, C, and E) or FIV(VSV) (B, D, and F) pseudotypes bearing a green fluorescent protein (GFP) marker gene. Seventy-two hours postinfection, GFP expression was quantified by flow cytometry. Infections were performed in the presence (filled symbols) or absence (open symbols) of medium supplemented with 2.5 μM CypA antagonists Cs (A and B), NIM811 (C and D), or Debio-025 (E and F) or their respective solvents dimethyl sulfoxide (DMSO) (Cs and NIM811) or ethanol (Debio-025). (G, H, and I) Sensitivity of HIV and FIV to Cs (G), NIM811 (H), and Debio-025 (I). TRIMCyp-expressing cells were incubated with inhibitor at 0, 0.5, 1.0, 1.5, or 2.0 μM prior to infection with HIV(VSV) or FIV(VSV) pseudotypes. Each point represents the mean of triplicate estimations.
FIG. 2.
FIG. 2.
Inhibition of viral replication by feline TRIMCyp and its reversal by CypA antagonists. CrFK (ID10) cells stably transduced with a retroviral vector bearing feline TRIMCyp (B and D) or vector only (A and C) were infected with FIV-Fca (Petaluma-F14 strain) (A and B) or FIV-Pco (CoLV strain) (C and D). Infections were performed in the presence or absence of medium supplemented with 2.5 μM CypA antagonist NIM811 or Debio-025 or their respective solvents, DMSO and ethanol (EtOH). Supernatants were collected and assayed for RT activity by nonisotopic RT assay (LentiRT; CavidTech, Sweden). CON, control. (E to H) Syncytium formation in CrFK cells infected with FIV-Fca. Cells expressing vector only (E and G) or TRIMCyp (F and H) and infected with FIV-Fca in the presence of Debio-025 (G and H) or ethanol solvent control (E and F) were fixed and stained at 10 days postinfection with 1.0% methylene blue-0.2% basic fuchsin in methanol. The arrows indicate small syncytia, magnified in the inset (H).
FIG. 3.
FIG. 3.
Restoration of viral replication in TRIMCyp-expressing cells by 2.0 μm NIM811 and Debio-025. CrFK (ID10) cells stably transduced with a retroviral vector bearing feline TRIMCyp (B and D) or vector only (A and C) were infected with FIV-Fca (Petaluma-F14 strain) (A and B) or FIV-Pco (CoLV strain) (C and D). Infections were performed in the presence or absence of medium supplemented with 2.0 μM NIM811 or Debio-025. Supernatants were collected and assayed for RT activity.
FIG. 4.
FIG. 4.
Growth of primary strains of FIV-Fca is blocked by feline TRIMCyp. (A and B) ID10 cells stably expressing feline TRIMCyp (TRIMCyp) or vector only (CON) and retransduced with vectors bearing either DSRed (− CD134) or a CD134-DSRed fusion (+ CD134) were infected with GL8 (A) or PPR (B), and viral replication was monitored by RT assay as for Fig. 2. (C to E) SIVmac infection is resistant to inhibition by feline TRIMCyp. ID10 control (CON) or TRIMCyp-expressing (TRIMCyp) cells were infected with SIVmac(VSV) GFP pseudotypes (D) and compared with FIV(VSV) (C) and HIV(VSV) (E) GFP pseudotypes. Infection was assessed 72 h postinfection by flow cytometry.

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