Lentiviral gene therapy against human immunodeficiency virus type 1, using a novel human TRIM21-cyclophilin A restriction factor

Abstract

TRIM5α (tripartite motif-containing protein-5, isoform α)-cyclophilin A fusion proteins are anti-human immunodeficiency virus (HIV) restriction factors that have evolved in certain nonhuman primates over millions of years and protect against HIV and related viruses. Restriction by TRIM5αCypA is potent and highly resistant to viral escape by mutation and, in combination with a suitable gene delivery platform, offers the possibility of novel therapeutic approaches against HIV. Here we report that lentiviral vector delivery of human mimics of TRIM5α-cyclophilin A (TRIM5CypA) fusion proteins afforded robust and durable protection against HIV-1, but resulted in downregulation of host cell antiviral responses mediated by endogenous TRIM5α. We found that substitution of TRIM5α RING, B-box, and coiled-coil domains with similar domains from a related TRIM protein, TRIM21, produced a novel and equally potent inhibitor of HIV-1. Both TRIM5CypA and TRIM21CypA inhibited transduction by HIV-1-derived viral vectors and prevented propagation of replication-competent HIV-1 in human cell lines and in primary human T cells. Restriction factor-modified T cells exhibited preferential survival in the presence of wild-type HIV. Restriction was dependent on proteasomal degradation and was reversed in the presence of the cyclophilin inhibitor cyclosporin. Importantly, TRIM21CypA did not disturb endogenous TRIM5α-mediated restriction of gammaretroviral infection. Furthermore, endogenous TRIM21 antiviral activity was assessed by measuring inhibition of adenovirus-antibody complexes and was found to be preserved in all TRIMCypA-modified groups. We conclude that lentivirus-mediated expression of the novel chimeric restriction factor TRIM21CypA provides highly potent protection against HIV-1 without loss of normal innate immune TRIM activity.

FIG. 1.

Lentivirus-mediated expression of TRIMCypA restriction factors. (A) Schematic representation of native TRIM5α showing RING, B-box2, coiled-coil, and B30.2 (PRYSPRY) domains. Substitution of the B30.2 domain with human cyclophilin A (CypA) produced TRIM5CypA. An analogous construct was derived from TRIM21 to produce TRIM21CypA. The transgene cassettes were cloned into a self-inactivating lentiviral vector under the control of the spleen focus-forming virus long terminal repeat (SFFV LTR) and linked to expression of EGFP by an internal ribosomal entry sequence (IRES). (B) Challenge of transduced CRFK cells with HIV-YFP in the presence or absence of cyclosporin (Cs). Flow cytometry of cells revealed high levels of YFP and EGFP coexpression in cells transduced with control vectors expressing EGFP alone, but cells transduced to express TRIM5CypA or TRIM21CypA, did not coexpress YFP (top), indicating highly specific restriction. Cs inhibited these restrictive effects (bottom). (C) Transduced CRFK cells expressing GFP alone or in combination with TRIM5CypA and TRIM21CypA were exposed to increasing MOIs of HIV-YFP. Flow cytometry for YFP expression was undertaken 72 hr later and showed efficient transduction of control populations at MOIs less than 10. Both TRIM5CypA and TRIM21CypA exhibited similar saturation effects, and required exposure to an MOI of 1000 to achieve complete transduction. (D) Western blotting of lentivirally transduced CRFK cells using anti-CypA antibody detected the presence of TRIMCyp species (TRIM5CypA 53kDa and TRIM21CypA 51kDa) in transduced populations (lanes 2 and 3) as well as native cyclophilin A (18 kDa) in control vector transduced cells (lane 1) and β-actin (bottom). (E) TRIM21CypA restriction of HIV reverse transcription was rescued by proteasomal inhibition by MG132, manifested as increased copies of GFP DNA. Rescue at this level was only partial, as GFP expression was still reduced compared with control (vector only) modified cells. Restriction was fully abrogated by treatment with Cs. (F) Flow cytometry showing inhibition of HIV-YFP infectivity in primary T cells transduced to express TRIM5CypA or TRIM21CypA, manifested as an absence of EGFP–YFP coexpression. NT, nontransduced control cells.

FIG. 1.

Lentivirus-mediated expression of TRIMCypA restriction factors. (A) Schematic representation of native TRIM5α showing RING, B-box2, coiled-coil, and B30.2 (PRYSPRY) domains. Substitution of the B30.2 domain with human cyclophilin A (CypA) produced TRIM5CypA. An analogous construct was derived from TRIM21 to produce TRIM21CypA. The transgene cassettes were cloned into a self-inactivating lentiviral vector under the control of the spleen focus-forming virus long terminal repeat (SFFV LTR) and linked to expression of EGFP by an internal ribosomal entry sequence (IRES). (B) Challenge of transduced CRFK cells with HIV-YFP in the presence or absence of cyclosporin (Cs). Flow cytometry of cells revealed high levels of YFP and EGFP coexpression in cells transduced with control vectors expressing EGFP alone, but cells transduced to express TRIM5CypA or TRIM21CypA, did not coexpress YFP (top), indicating highly specific restriction. Cs inhibited these restrictive effects (bottom). (C) Transduced CRFK cells expressing GFP alone or in combination with TRIM5CypA and TRIM21CypA were exposed to increasing MOIs of HIV-YFP. Flow cytometry for YFP expression was undertaken 72 hr later and showed efficient transduction of control populations at MOIs less than 10. Both TRIM5CypA and TRIM21CypA exhibited similar saturation effects, and required exposure to an MOI of 1000 to achieve complete transduction. (D) Western blotting of lentivirally transduced CRFK cells using anti-CypA antibody detected the presence of TRIMCyp species (TRIM5CypA 53kDa and TRIM21CypA 51kDa) in transduced populations (lanes 2 and 3) as well as native cyclophilin A (18 kDa) in control vector transduced cells (lane 1) and β-actin (bottom). (E) TRIM21CypA restriction of HIV reverse transcription was rescued by proteasomal inhibition by MG132, manifested as increased copies of GFP DNA. Rescue at this level was only partial, as GFP expression was still reduced compared with control (vector only) modified cells. Restriction was fully abrogated by treatment with Cs. (F) Flow cytometry showing inhibition of HIV-YFP infectivity in primary T cells transduced to express TRIM5CypA or TRIM21CypA, manifested as an absence of EGFP–YFP coexpression. NT, nontransduced control cells.

FIG. 1.

Lentivirus-mediated expression of TRIMCypA restriction factors. (A) Schematic representation of native TRIM5α showing RING, B-box2, coiled-coil, and B30.2 (PRYSPRY) domains. Substitution of the B30.2 domain with human cyclophilin A (CypA) produced TRIM5CypA. An analogous construct was derived from TRIM21 to produce TRIM21CypA. The transgene cassettes were cloned into a self-inactivating lentiviral vector under the control of the spleen focus-forming virus long terminal repeat (SFFV LTR) and linked to expression of EGFP by an internal ribosomal entry sequence (IRES). (B) Challenge of transduced CRFK cells with HIV-YFP in the presence or absence of cyclosporin (Cs). Flow cytometry of cells revealed high levels of YFP and EGFP coexpression in cells transduced with control vectors expressing EGFP alone, but cells transduced to express TRIM5CypA or TRIM21CypA, did not coexpress YFP (top), indicating highly specific restriction. Cs inhibited these restrictive effects (bottom). (C) Transduced CRFK cells expressing GFP alone or in combination with TRIM5CypA and TRIM21CypA were exposed to increasing MOIs of HIV-YFP. Flow cytometry for YFP expression was undertaken 72 hr later and showed efficient transduction of control populations at MOIs less than 10. Both TRIM5CypA and TRIM21CypA exhibited similar saturation effects, and required exposure to an MOI of 1000 to achieve complete transduction. (D) Western blotting of lentivirally transduced CRFK cells using anti-CypA antibody detected the presence of TRIMCyp species (TRIM5CypA 53kDa and TRIM21CypA 51kDa) in transduced populations (lanes 2 and 3) as well as native cyclophilin A (18 kDa) in control vector transduced cells (lane 1) and β-actin (bottom). (E) TRIM21CypA restriction of HIV reverse transcription was rescued by proteasomal inhibition by MG132, manifested as increased copies of GFP DNA. Rescue at this level was only partial, as GFP expression was still reduced compared with control (vector only) modified cells. Restriction was fully abrogated by treatment with Cs. (F) Flow cytometry showing inhibition of HIV-YFP infectivity in primary T cells transduced to express TRIM5CypA or TRIM21CypA, manifested as an absence of EGFP–YFP coexpression. NT, nontransduced control cells.

FIG. 1.

Lentivirus-mediated expression of TRIMCypA restriction factors. (A) Schematic representation of native TRIM5α showing RING, B-box2, coiled-coil, and B30.2 (PRYSPRY) domains. Substitution of the B30.2 domain with human cyclophilin A (CypA) produced TRIM5CypA. An analogous construct was derived from TRIM21 to produce TRIM21CypA. The transgene cassettes were cloned into a self-inactivating lentiviral vector under the control of the spleen focus-forming virus long terminal repeat (SFFV LTR) and linked to expression of EGFP by an internal ribosomal entry sequence (IRES). (B) Challenge of transduced CRFK cells with HIV-YFP in the presence or absence of cyclosporin (Cs). Flow cytometry of cells revealed high levels of YFP and EGFP coexpression in cells transduced with control vectors expressing EGFP alone, but cells transduced to express TRIM5CypA or TRIM21CypA, did not coexpress YFP (top), indicating highly specific restriction. Cs inhibited these restrictive effects (bottom). (C) Transduced CRFK cells expressing GFP alone or in combination with TRIM5CypA and TRIM21CypA were exposed to increasing MOIs of HIV-YFP. Flow cytometry for YFP expression was undertaken 72 hr later and showed efficient transduction of control populations at MOIs less than 10. Both TRIM5CypA and TRIM21CypA exhibited similar saturation effects, and required exposure to an MOI of 1000 to achieve complete transduction. (D) Western blotting of lentivirally transduced CRFK cells using anti-CypA antibody detected the presence of TRIMCyp species (TRIM5CypA 53kDa and TRIM21CypA 51kDa) in transduced populations (lanes 2 and 3) as well as native cyclophilin A (18 kDa) in control vector transduced cells (lane 1) and β-actin (bottom). (E) TRIM21CypA restriction of HIV reverse transcription was rescued by proteasomal inhibition by MG132, manifested as increased copies of GFP DNA. Rescue at this level was only partial, as GFP expression was still reduced compared with control (vector only) modified cells. Restriction was fully abrogated by treatment with Cs. (F) Flow cytometry showing inhibition of HIV-YFP infectivity in primary T cells transduced to express TRIM5CypA or TRIM21CypA, manifested as an absence of EGFP–YFP coexpression. NT, nontransduced control cells.

FIG. 1.

Lentivirus-mediated expression of TRIMCypA restriction factors. (A) Schematic representation of native TRIM5α showing RING, B-box2, coiled-coil, and B30.2 (PRYSPRY) domains. Substitution of the B30.2 domain with human cyclophilin A (CypA) produced TRIM5CypA. An analogous construct was derived from TRIM21 to produce TRIM21CypA. The transgene cassettes were cloned into a self-inactivating lentiviral vector under the control of the spleen focus-forming virus long terminal repeat (SFFV LTR) and linked to expression of EGFP by an internal ribosomal entry sequence (IRES). (B) Challenge of transduced CRFK cells with HIV-YFP in the presence or absence of cyclosporin (Cs). Flow cytometry of cells revealed high levels of YFP and EGFP coexpression in cells transduced with control vectors expressing EGFP alone, but cells transduced to express TRIM5CypA or TRIM21CypA, did not coexpress YFP (top), indicating highly specific restriction. Cs inhibited these restrictive effects (bottom). (C) Transduced CRFK cells expressing GFP alone or in combination with TRIM5CypA and TRIM21CypA were exposed to increasing MOIs of HIV-YFP. Flow cytometry for YFP expression was undertaken 72 hr later and showed efficient transduction of control populations at MOIs less than 10. Both TRIM5CypA and TRIM21CypA exhibited similar saturation effects, and required exposure to an MOI of 1000 to achieve complete transduction. (D) Western blotting of lentivirally transduced CRFK cells using anti-CypA antibody detected the presence of TRIMCyp species (TRIM5CypA 53kDa and TRIM21CypA 51kDa) in transduced populations (lanes 2 and 3) as well as native cyclophilin A (18 kDa) in control vector transduced cells (lane 1) and β-actin (bottom). (E) TRIM21CypA restriction of HIV reverse transcription was rescued by proteasomal inhibition by MG132, manifested as increased copies of GFP DNA. Rescue at this level was only partial, as GFP expression was still reduced compared with control (vector only) modified cells. Restriction was fully abrogated by treatment with Cs. (F) Flow cytometry showing inhibition of HIV-YFP infectivity in primary T cells transduced to express TRIM5CypA or TRIM21CypA, manifested as an absence of EGFP–YFP coexpression. NT, nontransduced control cells.

FIG. 2.

(A) TRIM5CypA and TRIM21CyA inhibit HIV replication. GHOST cells transduced and enriched for EGFP/TRIMCypA expression were infected with HIV-1 NL4-3 (Ba-L Env) and virus propagation was quantified by ELISA for p24 after 7 days. TRIM5CypA and TRIM21CypA groups had low levels of detectable p24 whereas in control cultures, HIV-1 had propagated as anticipated. NT, nontransduced control cells (n=3). (B) In cultures tracked for 28 days, we found that GHOST cells fully modified to express TRIM5CypA or TRIM12CypA did not support HIV propagation, and p24 levels were barely detectable by ELISA in these cultures. In contrast, nontransduced control populations had high p24 levels within 7 days of exposure to HIV, and cultures did not survive beyond this period, precluding further assessments (n=3). (C) Transduced primary T cells were enriched on the basis of CD4 and GFP coexpression and seeded in triplicate before challenge with HIV. Nontransduced T cells, or cells modified to express GFP alone, supported HIV replication and p24 was detectable within 5 days. In contrast, p24 levels in supernatant collected from TRIM5CypA- or TRIM21CypA-modified T cells were barely detectable, with no significant difference between TRIMCypA-modified populations (n=6, from two independent experiments). (D) SupT1 cells transduced to express TRIM5CypA or TRIM21CypA (linked to EGFP) were exposed to replication-competent HIV-1 in culture. Over a period of 14–28 days cells expressing HIV restriction factors increased in frequency, indicating preferential survival in the presence of replicating virus. (E) Survival advantage of TRIMCypA-modified populations was investigated in cultures in which approximately 20% of lentivirus-transduced cells were expressing GFP. Jurkat T cell viability was monitored by serial flow cytometry (with viability gating validated by LIVE/DEAD dye staining) and was found to be markedly reduced within 10 days of culture with HIV-1-nontransduced (NT) and GFP-transduced control populations. In cultures of TRIM5CypA- and TRIM21CypA-modified cells, the lowest viability mirrored the 20% proportion of cells expressing restriction factor and thus protected against HIV, and this rapidly improved over a further 7- to 14-day period, suggesting a strong survival advantage in the presence of replicating HIV.

FIG. 3.

TRIM5CypA but not TRIM21CypA inhibits endogenous TRIM5 function. (A) Loss of TRIM5-mediated restriction of the gammaretrovirus N-MLV was seen in cells modified to express TRIM5CypA, and this effect could not be rescued with interferon (IFN). TRIM21CypA-transduced cells retain their ability to restrict N-MLV-YFP, and as in the control groups, IFN-β augmented this effect, reducing YFP to background levels. (B) B-MLV was not restricted by either endogenous TRIM5α or the TRIMCyp proteins whereas (C) HIV was restricted by both TRIM5CypA and TRIM21CypA (n=3).

FIG. 3.

TRIM5CypA but not TRIM21CypA inhibits endogenous TRIM5 function. (A) Loss of TRIM5-mediated restriction of the gammaretrovirus N-MLV was seen in cells modified to express TRIM5CypA, and this effect could not be rescued with interferon (IFN). TRIM21CypA-transduced cells retain their ability to restrict N-MLV-YFP, and as in the control groups, IFN-β augmented this effect, reducing YFP to background levels. (B) B-MLV was not restricted by either endogenous TRIM5α or the TRIMCyp proteins whereas (C) HIV was restricted by both TRIM5CypA and TRIM21CypA (n=3).

FIG. 3.

TRIM5CypA but not TRIM21CypA inhibits endogenous TRIM5 function. (A) Loss of TRIM5-mediated restriction of the gammaretrovirus N-MLV was seen in cells modified to express TRIM5CypA, and this effect could not be rescued with interferon (IFN). TRIM21CypA-transduced cells retain their ability to restrict N-MLV-YFP, and as in the control groups, IFN-β augmented this effect, reducing YFP to background levels. (B) B-MLV was not restricted by either endogenous TRIM5α or the TRIMCyp proteins whereas (C) HIV was restricted by both TRIM5CypA and TRIM21CypA (n=3).

FIG. 4.

Endogenous TRIM21 restricts adenovirus in the presence of neutralizing antibody, and functions in TRIM5CypA- or TRIM21CypA-expressing cells. (A) HeLa cells stably knocked down for TRIM21 (T21 kd) or control cells expressing nontargeting shRNA (Control kd) were infected with adenovirus-GFP incubated with a neutralizing (Neut mAb) or isotype-matched control (Control mAb) antibody. The control shRNA-expressing cells efficiently neutralized adenovirus infection when neutralizing antibody was present. In contrast, HeLa cells depleted for TRIM21 were less efficient in neutralizing adenovirus infection in the presence of neutralizing antibody. (B) Confirmation that interferon-α induced TRIM21 expression, and this could be depleted by shRNAi targeting TRIM21 but not by control shRNAi. (C) After lentiviral transduction, TRIM5CypA- and TRIM21CypA-modified cells retained their ability to restrict adenovirus treated with neutralizing antibody, in the presence or absence of interferon-α, indicating intact endogenous TRIM21 activity in all populations. NT, nontransduced control cells (n=3).

FIG. 4.

Endogenous TRIM21 restricts adenovirus in the presence of neutralizing antibody, and functions in TRIM5CypA- or TRIM21CypA-expressing cells. (A) HeLa cells stably knocked down for TRIM21 (T21 kd) or control cells expressing nontargeting shRNA (Control kd) were infected with adenovirus-GFP incubated with a neutralizing (Neut mAb) or isotype-matched control (Control mAb) antibody. The control shRNA-expressing cells efficiently neutralized adenovirus infection when neutralizing antibody was present. In contrast, HeLa cells depleted for TRIM21 were less efficient in neutralizing adenovirus infection in the presence of neutralizing antibody. (B) Confirmation that interferon-α induced TRIM21 expression, and this could be depleted by shRNAi targeting TRIM21 but not by control shRNAi. (C) After lentiviral transduction, TRIM5CypA- and TRIM21CypA-modified cells retained their ability to restrict adenovirus treated with neutralizing antibody, in the presence or absence of interferon-α, indicating intact endogenous TRIM21 activity in all populations. NT, nontransduced control cells (n=3).

FIG. 4.

Endogenous TRIM21 restricts adenovirus in the presence of neutralizing antibody, and functions in TRIM5CypA- or TRIM21CypA-expressing cells. (A) HeLa cells stably knocked down for TRIM21 (T21 kd) or control cells expressing nontargeting shRNA (Control kd) were infected with adenovirus-GFP incubated with a neutralizing (Neut mAb) or isotype-matched control (Control mAb) antibody. The control shRNA-expressing cells efficiently neutralized adenovirus infection when neutralizing antibody was present. In contrast, HeLa cells depleted for TRIM21 were less efficient in neutralizing adenovirus infection in the presence of neutralizing antibody. (B) Confirmation that interferon-α induced TRIM21 expression, and this could be depleted by shRNAi targeting TRIM21 but not by control shRNAi. (C) After lentiviral transduction, TRIM5CypA- and TRIM21CypA-modified cells retained their ability to restrict adenovirus treated with neutralizing antibody, in the presence or absence of interferon-α, indicating intact endogenous TRIM21 activity in all populations. NT, nontransduced control cells (n=3).