Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha

Abstract

Mammalian cells express several factors that act in a cell-autonomous manner to inhibit retrovirus replication. Among these are the Friend virus susceptibility factor 1/lentivirus susceptibility factor 1/restriction factor 1 (Ref1) class of restriction factors, which block infection by targeting the capsids of diverse retroviruses. Here we show that lentivirus susceptibility factor 1 and Ref1 are species-specific variants of tripartite interaction motif 5alpha (TRIM5alpha), a cytoplasmic body component recently shown to block HIV-1 infection in rhesus macaque cells, and can indeed block infection by widely divergent retroviruses. Depletion of TRIM5alpha from human cells relieved restriction of N-tropic murine leukemia virus (N-MLV), and expression of human TRIM5alpha in otherwise nonrestricting cells conferred specific resistance to N-MLV infection, indicating that TRIM5alpha is Ref1 or an essential component of Ref1. TRIM5alpha variants from humans, rhesus monkeys, and African green monkeys displayed different but overlapping restriction specificities that were quite accurately predicted by the restriction properties of the cells from which they were derived. All TRIM5alpha variants could inhibit infection by at least two different retroviruses, and African green monkey TRIM5alpha was able to inhibit infection by no less than four divergent retroviruses of human, non-human primate, equine, and murine origin. However, each TRIM5alpha variant was unable to restrict retroviruses isolated from the same species. These data indicate that TRIM5alpha can confer broad innate immunity to retrovirus infection in primate cells and is likely to be an important natural barrier to cross-species retrovirus transmission.

Fig. 1.

Rhesus monkey and AGM TRIM5α variants inhibit HIV-1 infection when expressed in murine or feline cells. (A) Alignment of human (Hu.1), rhesus (Rh.1), and AGM TRIM5α protein sequences. The asterisks indicate the positions of amino acids that differ in Hu.2 (R136Q) and Rh.2 (S184L, W196R, and T307P) alleles of TRIM5α.(B) Inhibition of HIV-1 infection by TRIM5α_rh.1 in murine cells. MDTF cells that were either unmodified or stably expressing TRIM5α_rh.1 were either mock-infected or infected with an HIV-1-GFP vector, as indicated. GFP expression measured in the FL1-H channel is plotted against side scatter (SSC-H). The percentage of cells falling within a gate (R2) that includes infected (GFP-positive) cells and excludes >99.9% of uninfected cells is indicated. (C) MLV-GFP or HIV-1-GFP infection of unmodified feline CRFK cells, or CRFK cells expressing Fv1ⁿ or TRIM5α_rh.1, as indicated. (D) Effect of variant human, rhesus monkey, and AGM TRIM5α alleles, expressed in MDTF cells, on HIV-1 vector infection.

Fig. 2.

Human TRIM5α is necessary and sufficient to confer Ref1 activity. (A) Infection of MDTF cells that were unmodified (None) or expressing either of two human TRIM5α variants (hu.1 and hu.2) by N-MLV-GFP (filled bars) or B-MLV-GFP (open bars). The effects of Fv1ⁿ and Fv1^b expression on N-MLV and B-MLV infection is shown for comparison. (B) Silencing of TRIM5α_hu expression by using siRNA. HeLa cells were cotransfected with plasmids expressing HA-TRIM5α and GFP in the absence of siRNA (-) or in the presence of siRNAs targeting luciferase (Luc) or TRIM5α_hu (lanes A and B). (C) Infection of human (HeLa) cells with N-MLV or B-MLV after transfection with either no siRNA, the control-luciferase-specific siRNA, or the TRIM5-specific siRNA-A, as indicated. (D) Effects of control (Luc) and TRIM5-specific siRNAs (A and B) on N-MLV (filled bars) and B-MLV (open bars) infection of HeLa cells.

Fig. 3.

Non-human primate TRIM5α variants can inhibit infection by widely divergent retroviruses. (A) Infection of unmodified MDTF cells (None) or MDTF cells expressing AGM.1, rh.1, or rh.2 variants of TRIM5α by N-MLV (Left) and B-MLV (Right). (B–F) Infection of unmodified CRFK cells (None) or CRFK cells expressing hu.1, rh.1, or AGM.1 variants of TRIM5α, as indicated, by HIV-1 (B), SIV_MAC (C), HIV-1(SIV CA) (D), SIV_AGMTan (E), or SIV_AGMSab (F).

Fig. 4.

An equine retrovirus, EIAV, is inhibited by multiple human and non-human primate TRIM5α variants. (A) Titration of an EIAV-GFP vector on feline (CRFK), murine (MDTF), human (TE671 and HeLa), and non-human primate (FRhK4 and CV-1) cells. The percentage of GFP-positive cells as a function of inoculating EIAV–GFP dose, in CRFK infectious units (I.U.), is plotted. (B) Infection of unmodified CRFK cells (None) or CRFK cells expressing the indicated TRIM5α or Fv1 variants. (C) Infection of human (HeLa) cells with EIAV (filled bars) or N-MLV (open bars) after transfection with either no siRNA (None), the control-luciferase-specific siRNA (Luc), or the TRIM5-specific siRNA-A, as indicated.