Expression of HERV-K108 envelope interferes with HIV-1 production

Abstract

The human endogenous retroviruses (HERV)-K of the HML-2 group include full-length or near full-length elements encoding functional proteins, and are classified as type-1 or type-2 (type-1 has a deletion in the 5' end of the env gene). Because proteins of different retroviruses can interact, we hypothesized that HERV-K envelope (Env) could influence HIV-1 replication. Here we describe the negative effect of envelope expression of certain type-2 HERV-Ks on HIV-1 production. All HIV-1 and SIV strains tested were susceptible to various degrees to inhibition by the HERV-K108 envelope. We identified four residues within HERV-K108 Env as being critical to inhibit HIV-1 production. No inhibition was observed on EGFP expression, indicating that HERV-K Env does not affect general protein production. These findings demonstrate that envelope proteins from some endogenous retroviruses can limit production of exogenous lentiviruses such as HIV-1. Future studies will elucidate the mechanism mediating HIV-1 inhibition by HERV Envs.

Keywords: Endogenous retroviruses; Envelope; HERV-K; HIV-1.

Figure 1

A) Effect of different HERV-K envelopes on HIV protein production. Western blot of HEK 293T transfected with 3 different HERV-K envs belonging to the type-2 group (Kcon, K109 and K108) and 2 belonging to the type-1 group (K102 and K18) transfected alongside HIV-1 NL4-3. Because sequences belonging to the different envs have different protein expression efficiencies, we adjusted plasmid amounts in order to obtain comparable amounts of protein. The anti-HERV-K Env antibody HERM 1811-5 used throughout the paper recognizes the transmembrane domain (TM) on the C-terminus of HERV-K Env, which explains the detection of full length (FL) as well as of the processed TM portion of the Env protein. B) HERV-K108 Env does not interfere with HIV-1 transcription. 32 hours after transfection, quantitative RT-PCR was performed on RNA extracted from cells transfected with different amounts of either HERV-K108 env or empty plasmid control alongside 250ng of NL4-3 expressing plasmid. Data are normalized with the expression of house keeping gene Ribosomal Protein S11 (RPS11). The results are the averages of 3 experiments and error bars represent standard deviations. P values of 2-tailed T-Tests for each amount HERV-K108 or control are all not significant, confirming that there are no differences between the values obtained. C) No effect of HERV-K Rec on HIV-1 expression. Western blot of lysates of HEK293T transfected with increasing amounts of HERV-K rec HA alongside plasmids encoding HIV-1 NL4-3. Quantifications represent the average of 3 experiments and the error bars represent the standard deviation from the mean. D) No effect of HERV-K 108 Env expression on EGFP protein production. Western blot of HEK 293T cells transfected with different amounts of HERV-K108 alongside 100ng of EGFP plasmid. Quantifications represent the average of 3 experiments and the error bars represent the standard deviation from the mean. E) Effect of HERV-K 108 Env expression on MLV Gag and Ebola VP40 production. Western blot of HEK 293T cells transfected with different amounts of HERV-K108 env alongside MLVgag-pol and ZEBOV VP40 matrix expressing plasmids. Quantifications represent the average of 3 experiments for MLV and 5 experiments for ZEBOV, and the error bars represent the standard deviation from the mean.

Figure 2

A) Both lab-adapted and transmitter-founder strains of HIV-1 are sensitive to HERV-K108 Env. Western blot of lysates of HEK293T transfected with increasing amounts of HERV K108 env alongside plasmids encoding the following strains of HIV-1: NL4-3, LAI, WITO.c/2474 and CH040.c/2625. Quantifications represent the average of 3 independent experiments and the error bars represent the standard deviation from the mean. B) Different lentiviruses are sensitive to HERV-K108 Env. Western blot of lysates of HEK293T transfected with increasing amounts of HERV K108 env alongside plasmids encoding the following lentiviruses: HIV-1 NL4-3 Luc.HXB3, SIVcpz LB715, HIV-2 ROD10, SIVmac239. Quantifications are based on the average of four independent experiments and the error bars represent the standard deviation from the mean. FL stands for full-length and TM for transmembrane domain.

Figure 3

A) HERV-K108 Env expression inhibits HIV-1 virion production in cell culture supernatant. HIV-1 virion release in the tissue culture supernatant was measured 42h after transfection by ELISA for capsid p24. The results are represented as p24 production relative (%) to control, which was obtained by HIV-1 co-transfection with empty control vector. The results shown reflect the averages of three independent experiments and error bars represent standard deviations. B) HERV-K108 Env expression only minimally affects HIV-1 infectivity. TZM-bl reporter cell line was infected with 2.2ng of CA-p24 equivalents of HIV-1 produced in the presence of increasing amounts of HERV-K 108 env plasmid. Β-galactosidase was measured 44 hours after infection. The results presented are the averages of three independent experiments and error bars represent standard deviations. C) HERV-K108 Env expression in HeLa cells inhibits HIV-1 prduction. Western blot of lysates of HeLa cells transfected with increasing amounts of HERV K108 env alongside plasmids encoding HIV-1 LAI. Quantifications represent the average of 3 experiments and the error bars represent the standard deviation from the mean.

Figure 4

A) Schematic representation of HERV-K con and HERV-K108 envelope sequences. The positions where the residues differ between HERV-K108 and HERV-Kcon are identified by red circles. The black circle highlights the furin cleavage site. SP stands for signal peptide (shown as a blue sequence), SU stands for surface domain, TM for transmembrane (TM ectodomain shown blue sequence) and CTD stands for cyotoplasmic domain. B) and C) Effect of furin cleavage site mutagenesis on the interference on HIV by HERV-K108 Env. Disruption of furin cleavage site inhibits HERV-Kcon Env processing and increases its inhibitory effect on HIV production, while optimization of the furin cleavage site increases HERV-K108 Env processing but does not rescue HIV-1 production. FL stands for full-length and TM for transmembrane domain. D) Mutagenesis of all four residues that differ between K108 and Kcon rescues HIV-1 production. Western blot of lysates of HEK 293T co-transfected with 250ng of HIV-1 pLAI2, 100ng pcRV1-K rev and 60ng of HERV-K108 env versions harboring all possible combinations of the 4 mutant residues as well as HERV-Kcon env. Quantifications reflect the average of three independent experiments and the error bars represent the standard deviation from the mean. FL stands for full-length and TM for transmembrane domain.