EIAV encodes an accessory protein that antagonizes the host restriction factor equine tetherin

Abstract

Equine infectious anemia virus (EIAV) is an important model for the study of pathogenesis in lentiviruses. Studies of viral genome organization and replication mechanisms are fundamental to the understanding of virus pathogenicity. In this study, we identified an unique transcript from EIAV in vivo and in vitro by Sanger sequencing and Northern blotting. The transcript contains a complete open reading frame and has length 369 nt. We named the protein encoded by this transcript S4 and demonstrated its expression in EIAV-infected cells. An S4-deficient EIAV infectious clone displayed obviously impaired virion release and attenuated virus replication in vitro, demonstrating that S4 plays a role in the release step of EIAV. The host restriction factor tetherin has broad-spectrum antiviral activity and prevents the release of a wide range of enveloped viruses, including lentiviruses. Here, we demonstrated that S4 enhances the release of the EIAV-like particle by counteracting the equine tetherin (eqTHN). S4 interacts with the eqTHN and sequesters it within intracellular membrane compartments, attenuating eqTHN expression on the cell surface and thereby disrupting its antiviral activity. Further investigation revealed that S4 retains eqTHN in the endoplasmic reticulum and trans-Golgi network through impacting its anterograde transport to the cell surface and may interfere with the posttranslational modification of this membrane protein. Collectively, our findings uncover an accessory protein, S4, of EIAV and reveal its ability to promote virion release by antagonizing the antiviral activity of the host restriction factor tetherin.

Keywords: EIAV; lentivirus; tetherin; transcript; viral evasion.

Fig. 1.

A novel protein S4 was identified from EIAV. (A) Proviral organization and splicing pattern of the EIAV genome. The five fully spliced transcripts from EIAV identified by nested PCR (an upstream primer, p399; two downstream primers, p8144 and p8081) in previous studies are shown below the proviral organization. The numbers indicate the splice sites of each transcript. Exons are represented by solid lines. The horizontal arrows (p399 and p7603/p7583) show the positions of the primers used for the identification of the s4 transcript. (B) Sequence alignment of the s4 transcript and EIAV proviral genome (GenBank accession number: GU385365.1). The predicted s4 open reading frame (ORF) is highlighted in gray, and the predicted rev ORFs are underlined. The arrows indicate the start and stop codons. The intron in the processed s4 transcript is omitted and is indicated by a dotted line. (C and D) Identification of the s4 transcript sequence from lymph node, spleen, kidney, brain, heart, marrow, liver, and testis of horses infected with EIAV_LN40 (C) or from eMDMs infected with EIAV_DLV121 and EIAV_DLV34 (D)_. The primer pair p399 and p7603 was used to amplify the cDNAs of s4 in a first-round PCR after a reverse transcription assay. The primer pair p399 and p7583 was used in the second-round PCR, as indicated. (E) Identification of s4 transcript from EIAV-infected cells with Northern blot (NB) assay. The eMDMs were infected with EIAV_DLV121. Cell culture medium was used to mock-infect eMDMs as a negative control (NC). Total RNA was extracted at 96 h postinfection (hpi) for the NB assay, using the s4 gene as a probe. 18S RNA was used as a loading control. The experiment was repeated twice and a representative result is shown. (F) Identification of S4 from EIAV-infected cells with WB. The eMDMs were infected with EIAV_DLV121 at an MOI of 5. Cell culture medium was used for mock-infection of eMDMs as an NC. Cells were harvested at the indicated time points and were lysed before anti-p26 and anti-S4 antibodies were used for the detection of Gag and S4 expression. (*) indicates nonspecific bands. The experiment was repeated three times and a representative result is shown.

Fig. 2.

Inactivation of S4 inhibits the release of EIAV. (A) Inactivation of S4 reduced the release of EIAV. HEK293T cells were transfected with equivalent amounts (1 μg) of wild-type EIAV molecular clone (CMV3-8) or S4 inactivated EIAV molecular clone (CMV3-8∆S4). At 48 hpt, supernatants were harvested and were analyzed using reverse transcriptase (RT) activity. The data represent the means ± SD from three independent experiments. ***P < 0.001. (B) Similar to A, culture supernatants were ultracentrifuged to concentrate the viral particles after removing cell debris. Viral CA protein (p26) in the supernatant and Gag protein in the cell lysates were analyzed with WB using anti-p26 antibody. The experiment was repeated three times and one of the three independent experiments is shown. (C and D) S4 proteins expressed in trans rescued the release of S4-deficient EIAV. (C) HEK293T cells were cotransfected with CMV3-8∆S4 (1 μg) alone or in the presence of the GST-tagged S4 expression plasmids. At 48 hpt, virions released into the cell supernatant were analyzed with RT activity. The data represent the means ± SD from three independent experiments. (D) Similar to C, virions in the supernatant and cell lysates were and harvested analyzed with WB. Proteins in the viral and cell lysates were measured using anti-p26 and anti-GST antibodies. (E) Growth kinetics of the EIAV_CMV3-8 and the EIAV_CMV3-8∆S4. The equivalent titers of EIAV_CMV3-8 or EIAV_CMV3-8∆S4 were inoculated into eMDMs, and viral RNA was prepared from the supernatants at the indicated time points after infection and quantified using qRT-PCR. The results represent the means of two independent infections, and error bars represent the SD. (F) Replication of S4-deficient EIAV in eMDM is rescued by complementation of S4 in trans. EIAV_CMV3-8∆S4 with pseudotyped HIV-1 containing S4-HA or without S4-HA (as a specificity control) were inoculated into eMDMs, and viral RNA was prepared from the supernatants at the indicated time points after infection and quantified using qRT-PCR. The results represent the means of two independent infections, and error bars represent the SD.

Fig. 3.

S4 antagonizes eqTHN antiviral activity. (A) HEK293T cells were transfected with a codon-optimized EIAV Gag expression plasmid and human tetherin (huTHN) expression vectors in the presence of S4- or Vpu-expression plasmids. At 48 hpt, VLPs in the supernatant and proteins in cell lysates were detected using anti-p26 or indicated anti-tag antibodies. PNGase indicates PNGase F treatment for the evaluation of huTHN expression levels. (B) Knockdown or knockout of huTHN promotes the release of S4 inactivated EIAV in HEK293T cells. HEK293T cells were transfected with 50 nM huTHN-specific siRNA for 24 h to knockdown endogenous huTHN and then transfected with CMV3-8∆S4 or CMV3-8 (1 μg), and wild-type (WT) and huTHN knockout (KO) HEK293T cells were transfected with CMV3-8∆S4 or CMV3-8 (1 μg). At 48 hpt, cells were lysed and culture supernatants were ultracentrifuged to concentrate the virions. Subsequently, p26 in the supernatant and Gag protein in the cell lysates were analyzed with WB using anti-p26 antibody, and huTHN was analyzed using an anti-huTHN monoclonal antibody (ABclonal, Wuhan, China, A8839). si C, a scrambled siRNA, was used as a negative control; si 1, huTHN-specific siRNA. (C–E) S4 counteracts the inhibition of EIAV by eqTHN. HEK293T cells (C) or FDD cells (D) were transfected with a codon-optimized EIAV Gag expression plasmid and eqTHN expression vectors in the presence or absence of S4-expression plasmids. At 48 hpt, VLPs in the supernatant and proteins in cell lysates were detected using anti-p26 and indicated anti-tag antibodies. PNGase indicates PNGase F treatment for the evaluation of eqTHN expression levels. Expression of eqTHN was not detected by immunoblotting in D. (E) HEK293T cells were cotransfected with a fixed amount of Gag and eqTHN expression plasmids and the indicated amounts of S4-expression plasmids. At 48 hpt, proteins in the supernatant and cell lysates were detected using anti-p26 and indicated anti-tag antibodies. (F) Downregulation of endogenous eqTHN in eMDMs rescues EIAV_CMV3-8∆S4 replication. eMDMs were transfected with 50 nM eqTHN-specific siRNA (sieqTHN) or scrambled siRNA (as negative control, siNC) for 24 h to knockdown endogenous eqTHN and then infected with EIAV_CMV3-8∆S4. EIAV_CMV3-8 was used as a positive control. At the indicated time points, the viral RNA levels in the supernatant were determined as described in Fig. 2E. The results represent the means of two independent infections, and error bars represent the SD. (G) Immunoprecipitation analysis of eqTHN and S4. HEK293T cells were cotransfected with plasmids expressing eqTHN-Flag in the presence of the VR-S4-GST or VR-GST vector, cell lysates were prepared and immunoprecipitations were performed with anti-Flag beads and then analyzed using WB. (H and I) S4 relocalizes eqTHN from the PM to cytoplasmic compartments. (H) S4 and eqTHN expressing plasmids were transfected individually or together into HEK293T cells. At 48 hpt, cells were fixed, permeabilized, and costained with indicated anti-tag antibodies and then observed using confocal microscopy. Blue, cell nucleus; red, eqTHN tagged with Flag; green, S4 tagged with HA. Right: Histogram of the fluorescent intensity from the merged image obtained using the ZEISS ZEN Blue software. The colored wavy lines representing the fluorescence of the corresponding color show the colocalization of S4 and eqTHN. (I) HEK293T cells were transfected with eqTHN and S4 expressing plasmids or empty vector as a negative control. At 48 hpt, the cells were harvested and the mean fluorescence intensity (MFI) of eqTHN on the cell surface was examined with flow cytometry using an anti-Flag antibody followed by Alexa Fluor-488-conjugated goat anti-mouse antibody. The results are depicted as the mean of triplicate transfections with error bars representing the SD. All experiments were performed three times, and a representative result was shown. ****P < 0.0001.

Fig. 4.

The CD and ED are required for S4 to antagonize eqTHN. (A) HEK293T cells were transfected with a Gag expression plasmid and the eqTHN expression vectors in the presence or absence of S4 and S4 mutant expression vector. At 48 hpt, proteins in the supernatant and cell lysates were detected using anti-p26 or indicated anti-tag antibodies. PNGase indicates PNGase F treatment for evaluation of eqTHN expression levels. This experiment was performed two times and a representative result is shown. (B) Analysis of the effect of S4 mutants on the expression of eqTHN on the cell surface using flow cytometry. HEK293T cells were transfected with eqTHN and S4 or S4 mutant expression plasmids, or with plasmids expressing empty vector as a control. At 48 hpt, the cells were harvested and the MFI of eqTHN on the cell surface was examined with flow cytometry as described in Fig. 3I. MFI of eqTHN from triplicate transfections is shown with error bars representing the SD. One out of two independent experiments are shown. ****P < 0.0001; ns, not significant.

Fig. 5.

S4 affects eqTHN trafficking to the cell surface without promoting its internalization. (A) Internalization of eqTHN observed by confocal microscopy. HEK293T cells were transfected with eqTHN expression plasmid. At 48 hpt, cells were stained with an anti-Flag antibody followed by Alexa Fluor-488-conjugated goat anti-mouse antibody, and the antibody uptake was analyzed at 4 and 37 °C using confocal microscopy. (B) S4 antagonizes eqTHN without promoting its internalization. HEK293T cells were cotransfected with the eqTHN and S4 expression plasmids or empty vector. At 48 hpt, cell surface eqTHN was labeled with the anti-Flag antibody at 4 °C before incubating cells at 37 °C for the indicated time intervals to allow internalization. Cells were then incubated at 4 °C in the presence of the appropriate secondary antibody. MFI of eqTHN on the cell surface was examined with flow cytometry. The graph shows the relative levels of eqTHN at the cell surface (time 0 = 100%) and represents the loss of eqTHN-specific signal due to internalization. ns, not significant. MFI of eqTHN from triplicate transfections is shown with error bars representing the SD. One out of two independent experiments is shown. (C–E) S4 and Env affect eqTHN trafficking to the cell surface. An eqTHN expression plasmid was transfected into HEK293T cells alone (C) or with S4 (D) or Env (E) expression plasmids. At 48 hpt, cells were treated with pronase (0.05%) for 30 min (lines 2, 8, and 13), and untreated cells were used as a negative control (lines 1, 7, and 12). Cells were further incubated at 37 °C for 30, 60, and 180 min after the end of pronase treatment (lines 3 to 5, 9 to 11, and 14 to 16). Cells were then fixed, permeabilized, and costained with indicated anti-tag antibodies. Cell nucleus, blue; eqTHN, red; S4 or Env, green. In C, cells were further treated with brefeldin A (BFA, 10 μM) immediately after completion of the pronase treatment at 37 °C for 180 min as an intracellularly retained positive control (line 6). This experiment was performed three times. (F) Experiments were performed as in C–E, and the eqTHN expression on the cell surface was determined using flow cytometry as described in Fig. 3I. MFI values are presented for each gate. MFI of eqTHN from triplicate transfections is shown with error bars representing the SD. One out of three independent experiments is shown. ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig. 6.

S4 and Env have independent roles in eqTHN antagonism. (A) S4, Env, and gp90 expression constructs were transfected separately into HEK293T cells along with the Gag expression vector and the eqTHN expression vector, and then VLPs in the supernatant and proteins in cell lysates were determined using WB using anti-p26 or indicated anti-tag antibodies. PNGase indicates PNGase F treatment for evaluation of eqTHN expression levels. One of two independent experiments is shown. (B) HEK293T cells were transfected with eqTHN and either Env or S4 expression plasmids. At 48 hpt, the cells were either untreated (lanes II and IV) or treated with DMSO (lane I) or BFA (lane III) in DMSO for 12 h, after which they were harvested, and the proteins in the cell lysates were assessed with WB using indicated anti-tag antibodies. One of three independent experiments is shown.