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IL-27 posttranslationally regulates Y-box binding protein-1 to inhibit HIV-1 replication in human CD4+ T cells

Objectives: IL-27 is known as an antiviral cytokine that inhibits HIV, hepatitis C virus, and other viruses. We have previously demonstrated that, IL-27 posttreatment after HIV-infection inhibits viral replication in primary CD4+ T cells. Design: Here, we evaluated the anti-HIV effect of IL-27 pretreatment in CD4+ T cells from healthy donors prior to HIV infection with HIVNL4.3 or vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-luciferase virus (HIV-LUC-V). Methods: IL-27-treated CD4+ T cells were infected with HIVNL4.3 or HIV-LUC-V and assessed the anti-HIV effect. HIV infection was monitored by p24 antigen ELISA or luciferase assay. HIV fusion/entry and uncoating were determined by BlaM-Vpr assay and HIV fate of capsid and/or HIV Entry/Uncoating assay based on core-packaged RNA availability and Translation assay, respectively. HIV proviral copy number was determined by real-time PCR. Gene expression profile from IL-27-pretreated CD4+ T cells was determined using Genechip array. Posttranslational modification of global proteins from IL-27-pretreated CD4+ T cells was determined by a combination of 2-dimensional difference-in-gel-electrophoresis (2D-DIG), western-blot and protein mass spectrometry. Results: IL-27 pretreatment inhibited HIVNL4.3 and HIV-LUC-V infection in CD4+ T cells. HIV copy assay demonstrated that IL-27-treatment suppressed an early step of reverse transcription during HIV infection. A combination of 2D-DIG-electrophoresis and western blot assays demonstrated that IL-27-treatment induces a change in posttranslational modification of Y box binding protein-1 (YB-1). Overexpression of domain negative YB-1 mutants illustrated that a residue Lysine at 118 plays a key role in supporting HIV infection in CD4+ T cells. Conclusion: IL-27-pretreatment inhibits HIV-1 infection by suppressing an HIV-reverse transcription product formation/uncoating step by suppressing the acetylation of YB-1 in primary CD4+ T cells.


Introduction
We have previously reported that the pleotropic cytokine IL-27 inhibits HIV replication in primary CD4 þ T cells, monocyte-derived macrophages, and dendritic cells [1][2][3][4]. IL-27 is a heterodimeric cytokine composed of Epstein-Barr virus-induced gene 3 and IL-27p28 that belongs to the IL-6/IL-12 family of cytokines [5]. It is primarily secreted by macrophages and dendritic cells [6][7][8]. A recently published report indicated that plasma IL-27 levels in HIV patients were not significantly altered during HIV-1 infection [9], suggesting that IL-27 could potentially be used in anti-HIV therapy [10].
HIV utilizes host proteins to complete every step of its life cycle and replication process [11][12][13]. In addition, the host cells are equipped with proteins known as 'host restriction factors' that counter the HIV replication process. Cell survival and function are critically dependent on numerous cellular signals that are mostly mediated by posttranslational modifications (PTMs) of the proteins. Several studies report that PTMs of the host proteins either enhance or inhibit HIV replication [14][15][16][17], it is reported that Y-box binding protein-1 (YB-1), a DNA-binding and RNA-binding protein [18,19], serves as a host protein that stabilizes the genomic RNA of HIV-1 to promote HIV production in 293T cells [20]. It is also involved in HIV replication in Jurkat T cells [21]. In the current study, we investigated the anti-HIV mechanism of IL-27 in primary T cells. We now report, for the first time, that acetylated YB-1 is a key factor for HIV infection in primary T cells and that IL-27 regulates acetylation followed by HIV infection in T cells.

Material and methods
Participants were informed written consent prior to blood being drawn. All experimental procedures in these studies were approved by the National Cancer Institute at Frederick and National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board and performed in accordance with the relevant guidelines and regulations. Plasmids pNL4.3 [22] and pNL4.3.Luc [23,24] were obtained from the AIDS Reagents Program of the NIAID/National Institutes of Health (NIH) YB-1 expression vectors were constructed as follows: YB-1 wild-type and YB-1 lysinemutated genes were synthesized (Eurofins MWG Operon, Louisville Kentucky, USA) and cloned into pCMV5.1-Flag to obtain plasmid wild-type-YB-1-Flag and Mut-YB-1-Flag.

Pretreatment of CD4 R T cells with IL-27 and panabinostat
The PHA-stimulated CD4 þ T cells were pretreated with IL-27 prior to HIV infection. Briefly, the cells were cultured with either 20 U/ml of recombinant IL-2 (Roche, Mannheim, Germany) alone (control cells) or IL-2 with 100 ng/ml of recombinant IL-27 (R&D Systems, Minneapolis, Minnesota, USA) (treated cells) for 3 days in RP-10 at 2 Â 10 6 cells/ml. After incubation, the cells were washed with prewarmed RP-10 medium at 500 Â g for 5 min for three times, and then maintained in RP-10 with 20 U/ml of IL-2. Panabinostat (Selleckche, Houston, Texas, USA) treatment was performed as follows: PHA-stimulated CD4 þ T cells were pretreated with 35 nmol/l Panabinostat for 24 h. Following the pretreatment, CD4 þ T cells were treated with IL-2 or IL-2 with IL-27 for 3 days and then cells were washed as described above.

Flow cytometry analysis
Expression of CD4 þ , CXCR4, and CD3 on CD4 þ T cells was analyzed by flow cytometry. Cells were stained for 15 min at room temperature in PBS containing 2% bovine serum albumin with the following antibodies: Pacific Blue-conjugated anti-CD3 (Becton Dickinson, Franklin Lakes, New Jersey, USA), APC-Cy7-conjugated anti-CD4 þ (Becton Dickinson), and phycoerythrin (PE)-conjugated anti-CXCR4 (Becton Dickinson), all of which were performed following the vendor's protocol (Abcam, Cambridge, Massachusetts, USA). The cell preparations were analyzed with a FACSCalibur flow cytometer (Becton Dickinson). Positive and negative events were determined with matching isotype controls.
HIV-1 replication assay and HIV single-round infection IL-2 or IL-2/IL27-pretreated CD4 þ T cells were infected with HIV NL4.3 as previously described [2]. Half of the culture supernatants were replaced with fresh medium on day 3. Viral replication was gauged from p24 levels in culture supernatants using an HIV-1 p24 ELISA kit (PerkinElmer, Waltham, Massachusetts, USA). For singleround infection, CD4 þ T cells were incubated with HIV-LUC-V (100 ng/ml p24) for 2 h. Cells were washed and then cultured for 2 days in the presence of IL-2. Luciferase activity was measured with a Bright-Glo Luciferase Assay System (Promega, Madison, Wisconsin, USA).
HIV binding assay CD4 þ T cells were incubated with virus at 4 8C for 80 min to allow binding but not membrane fusion, as previously described [28], and then washed to remove unbound virus. Cells were lysed using RIPA buffer (Boston BioProducts, Ashland, Massachusetts, USA) to obtain the protein, which was followed by a HIV-1 p24 ELISA to assess the level of bound HIV.
HIV reverse transcription product assay Control and IL-27-pretreated CD4 þ T cells were infected with 100 ng/ml of pseudotyped HIV (DNase I-pretreated) for 2 h. Twelve hours postinfection, genomic DNA was isolated using a QIAamp DNA Blood Mini Kit (Qiagen). A total of 500 ng of genomic DNA was used for each reaction of qRT-PCR. Absolute copy number per 1 Â 10 6 cells were normalized by RNase P housekeeping gene (Applied Biosystems), as previously described [3].

2-Dimensional difference-in-gel-electrophoresis, two-dimensional gel electrophoresis, and western blot analysis
Whole-cell lysate protein samples from control and treated CD4 þ T cells were supplied to Applied Biomics (Hayward, California, USA) for identification of regulated global proteins by 2-dimensional differencein-gel-electrophoresis (2D-DIGE).
Whole-cell lysate proteins (100 mg) were subjected to 2D-PAGE analysis as described [30]. After separation by 2D-PAGE, the samples were transferred to a membrane for western blot analysis.

BlaM-Vpr assay for fusion and entry
The BlaM-Vpr entry/fusion assay was performed as previously described [26], and the fusion and entry activity in the samples were analyzed by flow cytometry using excitation at 410 nm and emission at 450/550 and 525/550 nm.
Immunoprecipitation and identification of posttranslational modification of Y box binding protein-1 Following nucleofection of CD4 þ T cells with 2 mg of wild-type-YB-1-Flag plasmid, cells were stimulated with PHA, followed by IL-2 or IL-2/IL-27 treatment for 3 days. Cells were harvested and lysed in IP lysis buffer (Thermo Fisher Scientific). A total of 500 mg of protein was used for immunoprecipitation, following the manufacturer's protocol (Sigma-Aldrich) using glycine/ Tris elution. The eluted protein was concentrated using methanol/chloroform precipitation, and samples were subjected to mass spectrometry (MC) analysis for identification of PTM.

Fate-of-capsid assay
The fate-of-capsid assay was performed as previously described [31], with 10 Â 10 6 control or IL-27-treated CD4 þ T cells infected with pseudotyped HIV-LUC-V virus (250 ng/ml) for 2 h, then washed and incubated for 12 h in RP-10 medium with IL-2. Infected CD4 þ T cells were harvested and resuspended in 2.5 ml of hypotonic lysis buffer (10 mmol/l Tris-Cl pH 8.0, 10 mmol/l KCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA), complete protease inhibitors (Roche) and a phosphatase inhibitor). After ultracentrifugation as previously described [31], the top layer above the sucrose cushion was collected as the 'soluble' fraction (1 ml), and, once the remaining sucrose was removed, SDS-PAGE loading dye was added to the tube to resuspend the 'pellet' fraction. Fractions were analyzed for capsid protein content by immunoblotting using a mouse monoclonal anti-HIV-1 p24 antibody (Abcam).
HIV Entry/Uncoating assay based on corepackaged RNA availability and translation assay The HIV EURT assay for HIV core protein uncoating was performed as previously described [27]. Briefly, control and treated CD4 þ T cells were infected with 500 ng/ml p24 HIV EURT virus for 2 h. Following infection, the cells were incubated in RP-10 medium with IL-2 for 12 h. Luciferase activity was measured with a Bright-Glo Luciferase Assay System (Promega).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5 software (San Diego, California, USA). Error bars indicate SDs or standard errors (SE) from means as noted. An unpaired Student's t test was used, and P values lower than 0.05 were considered significant.

IL-27 inhibits the early steps of HIV replication in CD4 R T cells
To determine which part of the HIV life cycle is suppressed in the IL-27-treated cells, we first compared HIV-1 NL4.3 binding to CD4 þ T cells. The amount of viral p24 on the cell surface was not altered by IL-27 pretreatment, indicating that HIV binds at the same intensity during infection in both control and treated CD4 þ T cells (Fig. 2a). Next, we assessed the impact of IL-27 on HIV internalization or fusion/entry into the host cells using the BlaM-Vpr fusion/entry assay [26]. No significant differences were observed between the control and treated cells in terms of CCF2-AM dye cleavage, an indicator of viral fusion/entry. Thus, IL-27 doesn't affect the fusion/entry step of HIV infection (Fig. 2b), suggesting that IL-27 treatment may affect the reverse transcription step.
To define IL-27's role, an early reverse transcription product (R-U5) and a late reverse transcription product (U5-Gag) were semi-quantitated using qPCR. IL-27 inhibited the early reverse transcription product by 72 AE 7.0% and inhibited the late reverse transcription product by 64 AE 1.0% (n ¼ 3) (Fig. 2c and d). In its natural state, HIV-1 provirus utilizes long terminal repeat (LTR) and Tat to mediate viral gene transcription. By cotransfecting the plasmid-encoding LTR-Luc and Tat [3], we were able to evaluate HIV promoter activity in control and IL-27-pretreated CD4 þ T cells. Pretreating CD4 þ T cells with IL-27 had no significant effect on viral transcription (n ¼ 3, P ¼ 0.15) (Fig. 2e). Thus, IL-27 pretreatment inhibits the reverse transcription step in the HIV-1 life cycle. IL-27 regulates the posttranslational modification HIV-1 utilizes host cell proteins as host-required factors to facilitate and complete its replication cycle. We hypothesized that IL-27 changes the expression of host factors in CD4 þ T cells. To identify the potential host factor involved in IL-27-mediated HIV-1 inhibition, we compared gene expression between control and IL-27treated T cells. A total of 25 000 genes were analyzed (Suppl. Fig. S2a, http://links.lww.com/QAD/B493), and only Radical S-Adenosyl Methionine Domain Containing Protein-2 (RSAD2) and Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) were upregulated more than four-fold in IL-27-treated cells. None of the genes were downregulated by more than four-fold. Real-time PCR confirmed increased gene expression in IL-27-treated CD4 þ T cells (Suppl. Fig.  S2b, http://links.lww.com/QAD/B493). The knockdown of either RSAD2 or IFIT3 by siRNA had no effect on the HIV-LUC-V infection (Suppl. Fig. S2c and d, http://links.lww.com/QAD/B493), suggesting that IL-27 may regulate an unknown host factor by PTM.

Y box binding protein-1 is an essential host-required factor for HIV infection
We sought the anti-HIV effects of the four posttranslationally regulated proteins using siRNA. Of the four, only siRNA-knockdown of YB-1 expression significantly inhibited the HIV-LUC-V infection ( Fig. 4a and b).
We further analyzed the effect of knocking down YB-1 in reverse transcription product formation following HIV-LUC-V infection. Figure 4c shows the knockdown efficiency of YB-1 siRNA in CD4 þ T cells (69% inhibition of YB-1 expression). This led to downregulation of the early and late reverse transcription product by 50 and 46%, respectively, compared with negative-control siRNA-transfected CD4 þ T cells (Fig. 4d and e). This result suggested that YB-1 is a host requirement factor that supports the HIV reverse transcription reaction in CD4 þ T cells during HIV replication.
Acetylated Y box binding protein-1 at lysine 118 residue is an essential host-required factor To identify the PTM of YB-1 responsible for HIV replication, we overexpressed Flag-tagged wild-type-YB-1 in CD4 þ T cells and cultured with IL-2 either alone or with IL-27. A 2D-gel western blot using an anti-Flag antibody demonstrated that overexpressed Flag-YB-1 exhibits a PTM pattern much like endogenous YB-1 ( Fig. 5a and b). Therefore, we used the cell lysate of the YB-1-Flag-overexpressed CD4 þ T cells for MC to determine PTM as described in the materials and methods.
Liquid chromatography-MC (LC/MS) revealed that lysine (K) residues K118, K137, and K264 in YB-1 were acetylated in control versus treated samples (Suppl. Fig.  S4a-c, http://links.lww.com/QAD/B493) indicating that the acetylated YB-1 plays a key role in HIV reverse transcription step and IL-27-mediated anti-HIV effect is led by inhibition of the YB-1 acetylation, which may be caused by either suppression of acetylation or enhancement of deacetylation of YB-1 by IL-27. To further confirm that posttranslational regulation of YB-1 (inhibition of acetylation) acetylation is a key event in IL-27-mediated anti-HIV effect in CD4 þ T cells, we pretreated the PHA-stimulated CD4 þ T cells with a panhistone deacetylase inhibitor, Panabinostat (35 nmol/l for 24 h at 37 8C), followed by IL-27 pretreatment, and infected the cells with HIV-LUC-V. Without Panabinostat treatment, IL-27 suppressed HIV replication to 47 AE 8.5% (n ¼ 3) and the inhibitor treatment restored the HIV replication to 84.5 AE 2.7% (n ¼ 3) (Suppl. Fig. S4d, http://links.lww.com/QAD/B493). This result illustrated that IL-27-mediated anti-HIV effect is led by induction of deacetylation of YB-1.
To identify the K residues responsible for HIV replication in YB-1, we designed YB-1 mutants with all three K residues mutated and three-mutants with a single K residue mutated to arginine (R). Following overexpression of wild-type-YB-1 and mutant-YB-1, CD4 þ T cells were infected with HIV-LUC-V. If YB-1 played a key role in HIV infection in T cells, it was assumed that overexpression of YB-1 would increase HIV infection. As expected, the overexpression of YB-1-wild-type-Flag increased HIV infection (Fig. 5c). Overexpression of YB-1 mutants lacking K137 (YB-1-DK137R-Flag) or K264 (YB-1-DK264R-Flag) also increased HIV infection. In contrast, overexpression of the YB-1 mutant lacking K118 (YB-1-DK118R-Flag) and YB-1-Mut-Flag (all three mutations: K118R/K137R/K264R) had no impact on HIV infection (n ¼ 3) (Fig. 5c). Overexpression of each YB-1 protein in CD4 þ T cells was confirmed by western blot (Fig. 5d). Acetylation of lysine-118 residue in YB-1 is therefore key in HIV replication. When this site was mutated to disable acetylation, HIV replication was set to the basal level in CD4 þ T cells.

IL-27 inhibits the uncoating of the HIV capsid core
After infection, the HIV core proteins must be disassembled in the host cell cytoplasm to release the reverse-transcribed viral genome. Therefore, the reverse transcription process is closely tied to the shedding of the viral capsid core proteins [33]. Since IL-27 inhibits the reverse transcription process in CD4 þ T cells, we further investigated the role of IL-27 during the uncoating process. If IL-27 inhibited the uncoating steps, the amount of intact core protein (stable core) in IL-27treated cells would be higher. The fate-of-capsid assay ( Fig. 5e and f, Suppl. Fig. S5a-d, http://links.lww.com/ QAD/B493) revealed that 34 AE 0.4% (n ¼ 3) and 59 AE 5.3% (n ¼ 3) of a total p24 proteins were in intact capsid core of control and IL-27-treated cells, respectively. This indicates that the capsid core shedding is 1.8fold (P < 0.05) lesser in IL-27-treated CD4 þ T cells. In the pelleted fraction (capsid core fraction), YB-1 protein was also detected, indicating either a potential interaction between capsid p24 and YB-1 protein during the viral capsid uncoating process or that YB-1 binds to HIV genomic RNA and gets pulled down in the pelleted fraction [20].
A recently established EURT using a reporter mRNAcoding luciferase [27] was applied to the control and IL-27-treated CD4 þ T cells. Then, we measured the firefly luciferase activity and observed a 35% reduction in the treated CD4 þ T cells (Fig. 5g, n ¼ 4), indicating that control CD4 þ T cells exhibit optimal uncoating and IL-27 treatment inhibits the uncoating process. We have 1826 AIDS 2019, Vol 33 No 12   (a and b) wild-type-Y box binding protein-1-Flag-overexpressed CD4 þ T cells were treated without (control) or with (treated) IL-27. We then performed 2dimensional-gel electrophoresis followed by western blot using anti-Flag antibody. (c) CD4 þ T cells were transfected with a series of expression vectors [wild-type-Y box binding protein-1-Flag, Y box binding protein-1-Flag lacking three acetylation sites (Y box binding protein-1-mutated-Flag), or Y box binding protein-1-Flag lacking a single acetylation site (Y box binding protein-1-DK118R-Flag, Y box binding protein-1-DK137R-Flag, Y box binding protein-1-DK264R)], and then infected with HIV-LUC-V. HIV infection activity was monitored by luciferase assay. Results show means AE SD from three independent assays. (d) Overexpression of Flag-Tag Y box binding protein-1 was detected by western blot using anti-Flag antibody. (e) Fate-of-capsid assay was performed as described in the materials and methods. Levels of HIV p24, Y box binding protein-1, and b-actin in pellet and supernatant fractions of control (C) and IL-27-treated CD4 þ T cells (T) were detected by western blot using antip24, Y box binding protein-1, or b-actin antibody, respectively. Input is the sample obtained before the separation of soluble and stable core proteins using sucrose separation. (f) ImageJ analysis of soluble and stable core p24 protein amounts in C and T was performed and normalized to the total confirmed that IL-27-pretreated CD4 þ T cells inhibit the uncoating process of the HIV capsid core protein, thereby inhibiting the reverse transcription product formation and, ultimately, suppressing HIV-1 replication.

Discussion
Our study has demonstrated that IL-27 promotes PHAactivated T cells into HIV-1-resistant T cells. IL-27 blocks HIV-1 infection post entry through a PTM of YB-1 that suppresses HIV core protein uncoating and inhibits the reverse transcription process. Silencing YB-1 in T cells strongly inhibits HIV-1 infection. Conversely, overexpression of YB-1 in T cells markedly increases the susceptibility of T cells to infection. Thus, our results suggest that modulating YB-1 by PTM or expression is an effective way to render human T cells resistant to HIV-1 infection (all assays and results are summarized in Suppl. Table 2, http://links.lww.com/QAD/B493).
Several reports have indicated that capsid uncoating is required for reverse transcription complex (RTC) formation/activation [34,35], which collectively supports the idea that HIV core uncoating precedes the reverse transcription process. In our study, the PTM of YB-1 by IL-27-pretreatment profoundly delayed capsid core shedding, thereby inhibiting proper RTC activation (which is observed by the diminished reverse transcription products followed by inhibition of HIV-1 replication). This supports the finding that uncoating precedes the reverse transcription process. The capsid uncoating was only 40% lesser in IL-27-treated CD4 þ T cells compared with control cells; however, the inhibition of HIV-1 replication in IL-27-treated CD4 þ T cells was 65-70% ( Fig. 1). This difference could be attributed to the fact that uncoating occurs early in the HIV-1 replication process, which eventually influences the reverse transcription, nuclear translocation, and integration. That has an additive effect on overall HIV-1 replication, which cumulatively amplifies the inhibitory effects. Therefore, IL-27-pretreatment inhibits HIV capsid core uncoating ( Fig. 5e-g) and overall HIV-1 replication. In addition, when using HIV EURT virus, luciferase activity fell to 65% in IL-27-treated cells (n ¼ 4) (Fig. 5g). Since we had observed low reverse transcription product (Fig. 4d and e) and inhibition of HIV-1 replication (Fig. 4b) in YB-1silenced CD4 þ T cells, we hypothesized that this leads to delayed HIV core uncoating in YB-1 k/d CD4 þ T cells. In YB-1-silenced CD4 þ T cells, increased accumulation of the pelleted p24 protein (1.42-fold) was observed compared with the negative siRNA control CD4 þ T cells (Suppl Fig. S5e, http://links.lww.com/QAD/B493) which is an indication that in the absence of YB-1, there is suppressed or delayed uncoating of HIV p24 capsid core. This finding further supports our hypothesis that YB-1 supports HIV replication by facilitating uncoating of HIV p24 core protein and in the absence of YB-1, uncoating is delayed or suppressed in infected CD4 þ T cells. To precisely define mechanism of acetylated YB-1 and role of residue of K118 on uncoating of core protein, we need further study.
HIV EURT assay detects an entered/uncoated HIV-1 that is based on the direct translation of a viral genomic RNA mimic coding a luciferase reporter that is delivered in target cells after viral-to-cellular membrane fusion in viral cores [27]. The HIV-1 entry/uncoating RNA genome coding Firefly luciferase (EU-repRNA-Luc) is added with the HIV-1 packaging sequence (C) upstream of Firefly luciferase followed by a poly A signal so that it behaves as a genomic viral RNA and gets encapsidated into virion particles. Upon virion maturation and Gag processing, this genome is contained within the viral core. Once the virion is delivered via infection into target cells, it is based on the two states of the capsid core (opened/ partially opened and closed). Where opened/partially opened, it allows the cellular ribosome machinery to translate luciferase protein, reflecting the uncoating process. If the core is closed, the cellular translation machinery, lacking access to EU-repRNA-Luc, will not translate luciferase protein. Thus, the luciferase assay reflects the uncoating process (the fate and status of viral core) in the HIV EURT assay. Using both the fate-ofcapsid assay and HIV EURT assay [27], we have confirmed that IL-27 pretreatment inhibits the HIV capsid core uncoating process; consequently, this could be the reason for reverse transcription product inhibition and, ultimately, HIV replication inhibition.
Recent data indicate that nucleotides required for reverse transcription are channeled into the core through electrostatic channels present in the capsid hexamer [36]. Thus, in control CD4 þ T cells, the dynamic capsid pores (with optimal uncoating process) may support nucleotide import and enhance the encapsidated DNA synthesis. Conversely, in treated CD4 þ T cells, the delayed uncoating process may disrupt the dynamics of nucleotide import, thereby inhibiting the reverse transcription products. However, this is just speculation, and a measure of incoming nucleotides into the capsid core is necessary to provide a stronger argument.
Rankovic et al. provided a strong argument that, during the reverse transcription process, the stiffness of the core increases immensely, which mechanically triggers capsid