Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity - PubMed (original) (raw)

Comparative Study

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity

Zuzana Keckesova et al. J Virol. 2006 May.

Abstract

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.

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Figures

FIG. 1.

FIG. 1.

Abrogation of cyclophilin A activity rescues HIV-1 infectivity in Old World monkey cell lines. HIV-1 GFP-encoding vectors were made using Gag from NL4.3, MVP5180, and NL4.3(Ba-L). Threefold serial dilutions were titrated onto Agm CV1 (A to C) and rhesus FRhK4 (D to F) cells. The cells were unmodified and untreated as a control (•), stably expressed siRNA to CypA (▵), or were treated with 5 μM CSA (○). NL4.3 GFP was also titrated onto Agm CV1 cells (G) or rhesus FrhK4 cells (H) stably expressing siRNA to CypA in the presence (▴) or absence (⧫) of 5 μM CSA. Virus input doses were measured in nanograms of reverse transcriptase as measured by ELISA. Results are representative of two independent experiments performed with two independent preparations of virus. (I) Western blot of extracts of CV1 and FRhK4 cells expressing reduced levels of CypA. Extracts from wild-type cells (wt) and cells expressing siRNA to CypA (KD) are shown. Actin blots are shown as a control for equal loading.

FIG. 2.

FIG. 2.

Reduction of TRIM5α protein levels renders HIV-1 infectivity insensitive to cyclosporine A. HIV-1 GFP-encoding vectors were made using Gag from NL4.3, MVP5180, or NL4.3(Ba-L). Threefold serial dilutions were titrated onto Agm CV1 cells (A to C) and rhesus FRhK4 cells (D to F). The cells were unmodified and untreated as a control (•), stably expressed siRNA to TRIM5 (▴), or stably expressed siRNA to TRIM5 and were treated with 5 μM CSA (⋄). To control for the reduction of TRIM5 expression, we show that MLV-N (▪) and MLV-B (□) GFP-encoding vectors have similar titers on Agm CV1 (G) or FRhK4 rhesus (I) cells expressing reduced levels of TRIM5α. MLV-N and MLV-B infectivities on untreated cells are shown as control Agm (H) and rhesus (J). Lentiviral input doses are measured in nanograms of reverse transcriptase as measured by ELISA. MLV input doses are shown as infectious units per milliliter on permissive feline CRFK cells. Results are representative of two independent experiments performed with two independent preparations of virus.

FIG. 3.

FIG. 3.

Expression of simian TRIM5α enables permissive feline CRFK cells to restrict HIV-1 in a cyclosporine-sensitive way. Serial dilutions of HIV-1 encoding GFP were titrated onto feline CRFK cells expressing Agm TRIM5α (A), rhesus TRIM5α (B), or unmodified CRFK cells as a control (C) in the presence of 5 μM CSA (⋄) or untreated as a control (⧫). Viral dose was measured by assay of reverse transcriptase (RT) by ELISA. (D) A fixed dose of HIV-1, chosen to infect around 1% of the target cells, was used to infect unmodified CRFK cells (•), CRFK cells expressing Agm TRIM5α (▴), or rhesus TRIM5α (○) in the presence of a serial dilution of CSA from 0 to 10 μM. The fold increase in infectivity after CSA treatment was measured and is plotted. Results are representative of two independent experiments performed with two independent virus preparations.

FIG. 4.

FIG. 4.

Cyclophilin A is not required for restriction of MLV or SIVmac by TRIM5α. (A) Threefold serial dilutions of MLV-N (○) or MLV-B (•) encoding GFP were titrated onto unmodified Agm CV1 cells as a control. (B) MLV-N (open symbols) and MLV-B (solid symbols) encoding GFP were titrated onto unmodified Agm CV1 cells in the presence of 5 μM CSA (▵) or onto Agm CV1 cells stably expressing siRNA to CypA (⋄). (C) Threefold serial dilutions of SIVmac encoding GFP were titrated onto unmodified, untreated Agm CV1 cells as a control (•), Agm CV1 cells in the presence of 5 μM CSA (▴), or Agm CV1 cells expressing siRNA to CypA (⧫). MLV input doses are shown as infectious units on permissive feline CRFK cells, and SIVmac input doses are shown as nanograms of reverse transcriptase (RT) as measured by ELISA. Results are representative of two independent experiments performed with two independent preparations of virus.

FIG. 5.

FIG. 5.

TRIM5α restricts HIV-1 independently of CypA binding to HIV-1 CA. Wild-type HIV-1 NL4.3 or MVP5180 (⧫) and their mutant CA G89V counterparts (⋄) were titrated onto Agm CV1 cells (A and B) and rhesus FRhK4 cells (C and D). The HIV-1 G89V mutant was also titrated onto cells expressing reduced levels of CypA protein (○). Wild-type HIV-1 (E and F) or their G89V mutant counterparts (G and H) were also titrated onto unmodified, permissive feline CRFK cells (▴), CRFK cells expressing rhesus TRIM5α (○), Agm TRIM5α (⋄), or owl monkey TRIM-Cyp (•). Virus input doses are measured in nanograms of reverse transcriptase, as measured by ELISA. Results are representative of two independent experiments performed with two independent preparations of virus.

FIG. 6.

FIG. 6.

Cyclosporine A reduces HIV-1 infectivity in human cells independently of TRIM5α. Threefold serial dilutions of wild-type HIV-1 NL4.3 (A and B) or HIV-1 CA G89V (C and D) were titrated onto TE671 cells stably expressing TRIM5 siRNA (A and C) or on unmodified cells as a control (B and D) in the presence (○) or absence (•) of 2.5 μM CSA. Threefold serial dilutions of MLV-N (⋄) and MLV-B (⧫) were titrated onto TE671 cells expressing TRIM5 siRNA (E) or unmodified TE671 cells (F). Effective reduction of TRIM5α expression is illustrated by equal MLV-N and MLV-B titers on cells treated with TRIM5 siRNA. HIV-1 input doses are measured in picograms of reverse transcriptase, as determined by ELISA. MLV input doses are shown as infectious units on permissive feline CRFK cells. Results are representative of two independent experiments performed with two independent preparations of virus.

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