Retroviral restriction factors Fv1 and TRIM5alpha act independently and can compete for incoming virus before reverse transcription

Abstract

The restriction factors Fv1 and TRIM5alpha provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5alpha-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5alpha for virus before reverse transcription. In human cells expressing Fv1(b), N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5alpha by Fv1. Our findings suggest that, like TRIM5alpha, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5alpha when expressed in human cells.

FIG. 1.

Expression of Fv1^b in human cells leads to additive restriction of MLV-N. (A and B) Serial dilutions of GFP-encoding MLV-N (⧫), MLV-B (▪), and MLV-NB (○) were titrated onto human TE671 cells expressing Fv1^b (TEB) (A) and unmodified TE671 cells (B). (C) Saturation experiment. TE671 (•) and TEB (⋄) cells were coinfected with a fixed dose of MLV-N encoding GFP and serial dilutions of MLV-N virus-like particles (N-VLPs). VLP dose in SC1 infectious units (i.u.) (x axis) is plotted against the fold increase in MLV-N GFP infectivity (y axis). (D) HIV-1 encoding GFP was titrated onto TE671 (•) and TEB (⋄) cells. Infected cells were enumerated 48 h later by FACS. Results are representative of at least two independent experiments performed with two independent preparations of virus. RT, reverse transcriptase.

FIG. 2.

MLV-N is less infectious but reverse transcribes more efficiently in human cells expressing Fv1^b. (A) Measurement of viral cDNA synthesis after restricted and unrestricted infection by TaqMan quantitative PCR. TE671, TE671 expressing Fv1^b (TEB), and TEB expressing reduced levels of TRIM5 (TEBT5KD) were infected in triplicate with equal doses of GFP-encoding MLV-N (black bars) or MLV-B (white bars). Six hours after infection, DNA was extracted from two samples and subjected to quantitative PCR in duplicate using primers and probe specific for GFP. (B) The third sample was analyzed by FACS 48 h after infection to measure permissivity. Errors are standard errors of the means. Results are representative of two independent experiments performed on two different TEB clones. SSC, side scatter.

FIG. 3.

Fv1^b is functionally independent of TRIM5α in human cells. (A) TE671 and TE671 cells expressing Fv1^b (TEB) were infected with a lentiviral vector carrying an shRNA against TRIM5 (T5) or a control shRNA against RFP. Unmodified cells were also infected as a control (Ct). Seventy-two hours later, cells were challenged with serial dilutions of MLV-N (black bars), MLV-B (white bars), or EIAV (dotted bars), and infectious titers per milliter are plotted. Errors are standard errors of the means. (B) TE671 (circles) and TEB cells (triangles) were infected with serial dilutions of MLV-N GFP in the absence (hollow symbols) or presence (filled symbols) of 2 μM arsenic trioxide (As₂O₃). Permissivity was measured 48 h after by FACS. (C) Saturation experiment. TE671 (circles) and TEB cells (triangles) were coinfected with a fixed dose of MLV-N encoding GFP and serial dilutions of EIAV VLPs. VLP dose (in nanograms of reverse transcriptase [RT]) (x axis) is plotted against the fold increase in MLV-N GFP infectivity (y axis). (D and E) The TRIM5δ splice variant was expressed in TE671 (D) and TEB cells (E) using a retroviral vector delivery system. Seventy-two hours later, cells were challenged with MLV-N or MLV-B encoding GFP. MLV-N and MLV-B infectivity on unmodified cells is also shown as a control. Infected cells were enumerated 48 h later by FACS. Results are representative of at least two independent experiments performed with two independent preparations of virus. i.u., infectious units. SSC, side scatter.

FIG. 4.

Fv1ⁿ is also functionally independent from TRIM5α. (A) TE671 cells expressing Fv1ⁿ (TEN) were infected with a lentiviral vector carrying an shRNA against TRIM5 (white bars) or left untreated (black bars). Seventy-two hours later cells were challenged with serial dilutions of MLV-N, MLV-B, or MLV-NB encoding GFP. Infection was analyzed by FACS, and data are presented as viral titers (infectious units [i.u.]/ml). Errors are standard errors of the means. (B) TEN cells were infected with serial dilutions of GFP-encoding MLV-N (circles) or MLV-B (triangles) in the absence (hollow symbols) or presence (filled symbols) of 2 μM arsenic trioxide (As₂O₃). Infected cells were measured 48 h after infection by FACS. (C and D) Saturation experiments. TEN cells were coinfected with a fixed dose of GFP-encoding MLV-N (circles) or MLV-B (triangles) and serial dilutions of either MLV-N (C) or EIAV (D) VLPs. VLP dose in SC1 i.u. or nanograms of reverse transcriptase (RT) (x axis) is plotted against the fold increase in MLV GFP infectivity (y axis). (E) The TRIM5δ splice variant was expressed in TEN cells using a retroviral vector delivery system. Seventy-two hours later cells were challenged with MLV-N or MLV-B encoding GFP. MLV-N and MLV-B infectivity on unmodified TEN cells is shown as a control. Infected cells were enumerated 48 h after infection by FACS. Results are representative of at least two independent experiments performed with two independent preparations of virus. SSC, side scatter.