Convergent evolution of antiviral machinery derived from endogenous retrovirus truncated envelope genes in multiple species

Abstract

Host genetic resistance to viral infection controls the pathogenicity and epidemic dynamics of infectious diseases. Refrex-1 is a restriction factor against feline leukemia virus subgroup D (FeLV-D) and an endogenous retrovirus (ERV) in domestic cats (ERV-DC). Refrex-1 is encoded by a subset of ERV-DC loci with truncated envelope genes and secreted from cells as a soluble protein. Here, we identified the copper transporter CTR1 as the entry receptor for FeLV-D and genotype I ERV-DCs. We also identified CTR1 as a receptor for primate ERVs from crab-eating macaques and rhesus macaques, which were found in a search of intact envelope genes capable of forming infectious viruses. Refrex-1 counteracted infection by FeLV-D and ERV-DCs via competition for the entry receptor CTR1; the antiviral effects extended to primate ERVs with CTR1-dependent entry. Furthermore, truncated ERV envelope genes found in chimpanzee, bonobo, gorilla, crab-eating macaque, and rhesus macaque genomes could also block infection by feline and primate retroviruses. Genetic analyses showed that these ERV envelope genes were acquired in a species- or genus-specific manner during host evolution. These results indicated that soluble envelope proteins could suppress retroviral infection across species boundaries, suggesting that they function to control retroviral spread. Our findings revealed that several mammalian species acquired antiviral machinery from various ancient retroviruses, leading to convergent evolution for host defense.

Keywords: antiviral; evolution; receptor; restriction factor; retrovirus.

Fig. 1.

CTR1 as the entry receptor for FeLV-D and Genotype I ERV-DCs. Viruses pseudotyped with the Envs shown in the x axis were tested in the following cell lines: (A) HEK293T cells, (B) MDTF cells, MDTF cells expressing human CTR1 (MDTF-huCTR1), and MDTF cells expressing feline CTR1 (MDTF-feCTR1) (Left). The expression of CTR1 was monitored by flow cytometry (Right). (C) Infection assay using HEK293T cells with endogenous CTR1 knockdown using siRNA. Expression of human CTR1 was analyzed by quantitative real-time RT-PCR and normalized by the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Left). Infective titers of the indicated LacZ-coding Env-pseudotyped viruses in HEK293T cells with CTR1 siRNA (Right). Enhanced GFP (EGFP) siRNA was used as a negative control. Comparisons with siEGFP control were performed using Student’s t test (*P < 0.01). (D) Env-pseudotyped viruses shown in A were tested in MDTF cells expressing human CTR2 (MDTF-huCTR2) (Left). Expression of CTR2 was monitored by RT-PCR (Right). The infectious units were assessed via log₁₀ β-galactosidase (LacZ)⁺ cells per milliliter of virus. Infective titers with SDs were means of three independent infection experiments. GI ERV-DCs, genotype I ERV-DCs; GIII ERV-DCs, genotype III ERV-DCs.

Fig. 2.

Refrex-1 inhibition of ancestral virus infection via CTR1. (A) Schematic representation of the structures of Refrex-1 and reconstructed ancestral Env proteins (ERV-DC7rec and ERV-DC16rec). Env genes of ERV-DC7rec and ERV-DC16rec were reconstructed by the removal of stop codons (triangles) and amino acid substitutions (arrows) based on ERV-DC7 and ERV-DC16 Env sequences, respectively. The amino acid substitutions were R407G, I427N, and T429A in ERV-DC7rec, and D431Y in ERV-DC16rec. PRR, proline-rich region; Sp, signal peptide. The number of amino acids (aa) is indicated on the right. (B) Infection of MDTF-feCTR1, MDTF-huCTR1, and MDTF cells by LacZ-coding Env-pseudotyped ERV-DC7rec and ERV-DC16rec viruses. The y axis indicates infectious units per milliliter of supernatant. (C) Dose-dependent inhibitory effect of Refrex-1 on viral infection. Supernatants of HEK293T cells transfected with expression vectors encoding ERV-DC7 Refrex-1 (blue), ERV-DC16 Refrex-1 (orange), or empty vector (Mock; gray) were diluted with medium; 250 µL of diluted supernatant was added to the culture when MDTF-feCTR1 or MDTF-huCTR1 cells were infected with ERV-DC7rec or ERV-DC16rec pseudotyped viruses. Infection titers with SDs are means of three independent infection experiments. Comparisons with Mock control were performed using Student’s t test (*P < 0.01).

Fig. 3.

Infection of primate Env-pseudotyped viruses belonging to FeLV-D interference groups. Infection of LacZ-coding primate Env-pseudotyped viruses in (A) HEK293T and CRFK cells and (B) MDTF-feCTR1, MDTF-huCTR1, and MDTF cells. (C) Infection of HEK293T cells and HEK293T cells preinfected with FeLV-D (ON-T), ERV-DC14, ERV-DC10, or FeLV-B by Env-pseudotyped viruses indicated in the x axis. Comparisons with 293T control were performed using Student’s t test (*P < 0.01). The y axis indicates infectious units per milliliter of virus. Infective titers with SDs are means of three independent infection experiments.

Fig. 4.

Effects of Refrex-1 and primate soluble Env proteins on retroviral infection. (A) Supernatants were collected from HEK293T cells transfected with expression vectors encoding ERV-DC7 Refrex-1, ERV-DC16 Refrex-1, or empty vector (Mock); HEK293T cells preincubated with the supernatant were then infected with pseudotyped viruses indicated in the x axis. (B) Schematic representation of Env structures. Number of amino acids (aa) is indicated on the right side. (C) Immunoprecipitation (IP) and Western blotting (WB) with anti–c-Myc antibody of supernatants from HEK293T cells transfected with indicated plasmids. Asterisks indicate truncated Env proteins. (D) Supernatants were collected from HEK293T cells that had been transfected with expression vectors encoding primate truncated Env proteins or empty vector (Mock); HEK293T cells preincubated with the supernatant were then infected with Env-pseudotyped viruses indicated in the x axis. The bar colors correspond to the truncated Env protein contained in the supernatant. The infectious units were assessed via log₁₀ LacZ⁺ cells per milliliter of virus. Infection titers with SDs are means of three infection experiments. Comparisons with Mock control were performed using Student’s t test (*P < 0.01).