Production of SIV in macrophages and CD4⁺ T cells

To produce isogenic SIV in CD4⁺ T cells and macrophages, it was imperative to employ a molecularly cloned SIV variant capable of robust replication in both cell types. Our choice for this purpose was a SIVmac239 variant, specifically SIVmac239/316 Env, characterized by nine amino acid substitutions within Env in comparison to the parental strain [22,23]. These substitutions facilitate efficient utilization of CCR5 in the absence of or at very low levels of CD4 expression [24], a condition observed in rhesus macaque macrophages [25]. For the preparation of SIVmac239/316 Env stocks, CD4⁺ T cells and macrophages were generated from rhesus macaque peripheral blood mononuclear cells (PBMCs) and their identity was confirmed by analysis of cell surface marker expression via flow cytometry (S1 Fig). Subsequently, CD4⁺ T cell and macrophage cultures were infected with SIVmac239/316 Env, input virus was removed, and the culture supernatants harvested over a two-week period. Supernatants containing more than 10 ng p27/ml were pooled to generate SIV stocks from macrophages (M-SIV) or CD4⁺ T cells (T-SIV). The resulting M-SIV and T-SIV stocks featured a p27-concentration of 7.9 and 52.2 ng/ml, RNA genome copies of 2.98 x 10⁷ and 2.13 x 10⁸/ml, and a TCID₅₀ of 80.353 and 28.184, respectively. Finally, sequencing of env, which was amplified through reverse transcriptase-polymerase chain reaction (RT-PCR) from the culture supernatants, provided confirmation that no genetic mutations were introduced into M-SIV or T-SIV throughout the in vitro propagation process.

The quantity of oligomannose-type and certain complex N-glycans of gp120 differs between M-SIV and T-SIV

The glycan shield of both HIV and SIV plays a pivotal role in immune evasion and mediating lectin-dependent interactions with immune cells. It is plausible that these functions could be modulated by cell type-specific alterations in the glycosylation pattern. To determine whether M-SIV and T-SIV differed in the N-glycosylation of the viral Env protein, we subjected concentrated virions to enzymatic digestion using endoglycosidase H (Endo H), which selectively removes oligomannose-type and certain hybrid-type N-glycans, and peptide-N-glycosidase F (PNGase F), which eliminates all N-linked glycans. The band shift upon Endo H digestion was more pronounced for T-SIV than M-SIV suggesting that T-SIV gp120 is adorned with a higher proportion of oligomannose-type glycans compared to M-SIV gp120 (Fig 1B). These findings align with previously published data [15,19,21] but do not offer insights into the specific structures of the differentially incorporated N-glycan species.

To address the latter question, we performed N-glycan analytics of T-SIV and M-SIV gp120 by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF). Further, the identity of the proteins subjected to N-glycan analytics was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The xCGE-LIF fingerprints of N-glycans derived from M-SIV and T-SIV gp120 show considerable differences in the relative intensities of the detected peaks suggesting quantitative differences in the incorporated glycan species (Figs 1C and S2).

A relative quantification of N-glycan signal intensities was performed. For this, the total peak intensity (tpi) of a peak was allocated to all N-glycan groups of the analysis, for which the assigned single or multiple glycan species met the criteria. This analysis revealed the following: T-SIV exhibited increased relative levels of oligomannose structures on gp120 compared to M-SIV (Fig 1D, 16.7% vs. 9.6% of tpi); in alignment with the results from glycosidase digestion and subsequent western blot analysis (Fig 1B). Additionally, profiles of complex-type N-glycans differed between the two viruses (Fig 1E–1G). M-SIV gp120 displayed more extensive branching of complex glycans, with a higher proportion featuring four antennae rather than three, in comparison to T-SIV (23.2% vs. 17.5% tpi) (Fig 1E). An overall assessment of fucose, sialic acid, and LacdiNAc content revealed that T-SIV gp120 harbors increased levels of N-glycan species carrying fucose (34 vs. 29.8 tpi) and sialic acid (51.7 vs. 48.8 tpi) compared to M-SIV; while the latter exhibited more LacdiNAc (11.8 vs. 7.4 tpi) containing structures (Fig 1F). A more detailed analysis of the complex-type glycan species revealed a more nuanced pattern: M-SIV complex glycans tended to feature either both fucose and sialic acid residues (23.8 (M-SIV) vs. 18.6% tpi (T-SIV)) or neither (8.7 (M-SIV) vs. 4.6% tpi (T-SIV)). Differently, T-SIV complex glycans tended to have either fucose (12.5 (T-SIV) vs. 5.7% tpi (M-SIV)) or sialic acid (33.1 (T-SIV) vs. 25% tpi (M-SIV)) but not both (Fig 1G). Our study revealed for the first time that despite an overall similar N-glycome, M-SIV and T-SIV gp120 differ in their oligomannose-type N-glycan content and complex N-glycan profiles, although due the use of a single pooled virus stock for M-SIV and T-SIV throughout the whole study our analysis was insufficiently powered to determine whether these differences were statistically significant.

M-SIV is better equipped for both direct and indirect viral spread compared to T-SIV

The revelation that M-SIV and T-SIV exhibit disparities in glycan coat composition raised the question of whether the differential origin of M-SIV and T-SIV is also linked to variations in other SIV characteristics pertinent to viral dissemination. To investigate this possibility, our initial focus was on viral infectivity. When TZM-bl indicator cells were exposed to viruses normalized for p27-capsid content, it became evident that M-SIV exhibited significantly higher infectivity than T-SIV (Fig 2A, left panel, 2-way ANOVA, p = 0.03). However, both viruses exhibited in vivo infectivity and replicated to comparable levels (S3 Fig), and when we equalized for the infectivity of M-SIV and T-SIV stocks, the infection efficiency on TZM-bl cells was found to be similar (Fig 2A, right panel). These results underscore that the infectivity of M-SIV per ng capsid antigen surpasses that of T-SIV, confirming the findings previously obtained for SIV [19] and dual-tropic HIV [20].

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Fig 2. M-SIV is more infectious and better transmitted by cellular lectins than T-SIV.

A) TZBM-bl indicator cells were exposed to equal volumes of M-SIV and T-SIV stocks, normalized for p27-capsid protein (left panel) or infectious units (right panel). Following virus removal at 5–6 hours post-infection, β-galactosidase activity was measured in cell lysates 72 hours post-infection. The grand mean of three experiments performed in triplicates (left panel) or quadruplicates (right panel) are shown with the standard error of the mean (SEM). Statistical significance between datasets determined by two-way ANOVA (*, p ≤ 0.05). B-C) B) M-SIV and T-SIV normalized for equal amounts of p27 were concentrated, resolved using SDS-PAGE, and analyzed by western blot for gp120 and p27. Consistent outcomes were observed across three independent experiments. C) The software ImageJ [55] was utilized to quantify gp120 and p27 signal intensities obtained in B). The gp120 signal per p27 signal ratio was calculated, and values were plotted, with M-SIV set as 100%. Presented is the grand mean with SEM. Paired t-test was applied to assess statistical differences between groups (**, p ≤ 0.01). D) M-SIV and T-SIV normalized for comparable infectivity on CEMx173 R5 target cells were incubated with Raji cells expressing no additional lectin, dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN), DC-SIGN related protein (DC-SIGNR) or Langerin. Unbound virus was removed and the transmitter cells were co-cultured with CEMx174 R5 target cells. Infection of target cells was detected by the measurement of luciferase activity in cell lysates 72 h post infection. Direct infection of CEMx174 R5 cells served as positive control, while negative controls consisted of Raji cells incubated without target cells. The signals obtained for the transinfection using transmitter cells expressing no lectin were subtracted as background signal. The grand mean of three independent experiments conducted in triplicates with SEM is depicted. Statistical differences between M-SIV and T-SIV for each transmitting lectin were calculated by t-test (*, p < = 0.05; ***, p < = 0.001). C.p.s.: counts per second.

https://doi.org/10.1371/journal.ppat.1012190.g002

Glycosylation also affects HIV Env incorporation into viral particles [26]. To clarify whether the increased M-SIV infectivity in comparison to T-SIV could be attributed solely to disparities in Env incorporation, we subjected p27-capsid normalized quantities of both M-SIV and T-SIV to western blot analysis to assess their gp120 and p27 content, and quantified the signal using the software ImageJ (Fig 2B and 2C). A visual examination already revealed that the less infectious T-SIV exhibited lower levels of gp120 incorporation compared to the more infectious M-SIV (Fig 2B). Following quantification and subsequent normalization of the gp120 signal to the p27-capsid signal, the data indicated a noteworthy 62% reduction in Env incorporation for T-SIV when compared to M-SIV (Fig 2C, t-test, p = 0.0051). While this effect is significant, the disparities in infectivity between the two viruses (Fig 2A, left panel, 55-fold at 6 ng p27/ml) surpasses the differences in Env incorporation. More research is needed to confirm a linear relationship between Env incorporation and SIV infectivity, however, these data imply a broader impact of the virus-producing cell on SIV infectivity beyond its influence on Env incorporation.

Lectins modulate HIV and SIV transmission to susceptible cells in vitro [7,27–29], a process possibly relevant for early viral spread after transmission in vivo [30]. Since both M-SIV and T-SIV were found to be infectious in rhesus macaques via the rectal route (S3 Fig), the question arose whether the cell type-dependent differences in these viruses influence their engagement of lectin receptors. Specifically, the interactions of M-SIV and T-SIV with the lectins with potential positive (DC-SIGN [7], DC-SIGN related protein (DC-SIGNR) [29]) or negative (Langerin [28]) influence on HIV/SIV viral spread were investigated. For this purpose a previously published transmission assay was employed [31]. CEMx174 R5 target cells were infected in two ways: directly with either virus, normalized for equal infectivity, or indirectly via co-culture with Raji transmitter cells bearing either no lectin or the aforementioned lectins (Fig 2D). While direct infection of target cells confirmed the equivalent infectivity of M-SIV and T-SIV, and co-culturing the virus with transmitter cells in the absence of target cells yielded only background signals, a significant difference emerged. M-SIV was more efficiently transferred to target cells by DC-SIGN compared to T-SIV (t-test, p = 0.01). This observation is consistent with previous findings related to the transmission of dual-tropic HIV produced in macrophages versus CD4⁺ T cells through DC-SIGN [20]. Similarly, DC-SIGNR and Langerin preferentially transferred M-SIV in comparison to T-SIV (t-test, p = 0.0001, 0.0003). These findings suggest that SIV replication in macrophages produces viral particles with distinctive characteristics that confer a clear advantage in direct and possibly indirect viral spread via lectins compared to viruses originating from CD4⁺ T cells.

Host cell-dependent features of SIV define the sensitivity to carbohydrate binding agents (CBAs) and neutralizing antibodies

CBAs bind glycan structures, such as those present on viral envelope proteins. Within the realm of antiviral activity against HIV, CBAs encompass a range of compounds including lectins, certain glycan-binding broadly neutralizing antibodies, and the nonpeptidic antibiotic Pradimicin A [32]. Consequently, next to non-glycan dependent broadly neutralizing antibodies, CBAs are viewed as promising biomedical treatments for preventing the transmission of HIV [32,33]. Due to the observed differences in N-glycomes between M-SIV and T-SIV, it was plausible that host cell-derived features of SIV Env might influence the effectiveness of CBAs and neutralizing antibodies. To explore this hypothesis, we examined the ability of CBAs to inhibit SIV infection of TZM-bl cells, the standard indicator cell line for neutralization assays in the field of HIV research [34]. In these experiments, we focused on two glycan species groups: oligomannose-type glycans and core fucosylated complex-type glycans, which were incorporated in different quantities into M-SIV and T-SIV gp120 (Fig 1D). In these experiments, we included HIV-1 Env as a reference protein and the vesicular stomatitis virus glycoprotein (VSV-G), which contains only two N-glycosylation signals per protomer [35] and thus should be non- or little sensitive to inhibition by CBA. VSV-G further differs from HIV/SIV Env by mediating fusion with the endosomal and not the plasma membrane [36]. In our experiments, the pre-treatment with ulex europaeus agglutinin (UEA), a lectin against core fucose, did not interfere with the infection of TZM-bl cells by controls (HIV-1 or VSV-G pseudotyped viruses) or SIV (Fig 3A) as expected [37]. As anticipated, HIV-1 was highly sensitive to inhibition by mannose-specific lectins cyanovirin-N (CV-N, target: Manα1–2) or galanthus nivalis agglutinin (GNA, target: Manα1–3, Manα1–6), while there was significantly less inhibition of infection by pseudotypes bearing the minimally glycosylated protein VSV-G [38,39]. Consistent with the western blot and xCGE-LIF data (Fig 1B and 1D), T-SIV with its apparently higher oligomannose content in gp120 was more strongly inhibited than M-SIV in a concentration-dependent manner (Fig 3A), and these differences were statistically significant (Fig 3B, t-test, CV-N: p = 0.0032, GNA: p = 0.008). Thus, the producer cell type can influence SIV susceptibility to inhibition by mannose-specific CBAs.

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Fig 3. T-SIV is more sensitive to inhibition by mannose-specific lectins, while M-SIV is more efficiently neutralized by sera.

A) Infectivity-normalized M-SIV, T-SIV, HIV-1 NL4-3 and env-defective HIV-1 NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) were preincubated with the indicated concentrations of ulex europaeus agglutinin (UEA), cyanovirin-N (CV-N), or galanthus nivalis agglutinin (GNA), before addition to TZM-bl indicator cells. Virus was removed at 5–6 h post infection and β–galactosidase activity was measured in cell lysates at 72 h post infection. Presented are the grand mean values with SEM normalized to lectin-free conditions from two independent experiments conducted in triplicates for all lectin concentrations. B) Plotted are the results obtained in A) for M-SIV and T-SIV using a lectin concentration of 100 μg/ml. Statistical differences between M-SIV and T-SIV were calculated for each lectin using a paired t-test (**, p ≤ 0.01). C) Infectivity-normalized M-SIV, T-SIV, and HIV-1 NL4-3 were preincubated with sera from SIVmac239-infected rhesus macaques at varying dilutions prior to infection of TZM-bl indicator cells. Incubation of virus with medium alone served as negative control. Virus removal occurred at 5–6 hours post-infection, with β-galactosidase activity measured in cell lysates at 72 hours post-infection. Shown is the grand mean with SEM from two independent experiments conducted in triplicates at a serum dilution of 1:20,000. Infection in the absence of serum was normalized to 100%. D) The titration curve for serum 3 from C) is presented as grand mean with SEM. E) Data for sera 1–4 from C), all at a 1:20,000 dilution, are directly compared. Paired t-test was applied to assess differences in means between groups (*, p ≤ 0.05).

https://doi.org/10.1371/journal.ppat.1012190.g003

To investigate whether cell type-dependent differences in SIV impact antibody neutralization effectiveness, we conducted infections of TZM-bl cells with HIV, M-SIV, and T-SIV in the presence of neutralizing sera. These sera were obtained from rhesus macaques infected with SIVmac239, the parental strain of SIVmac239/316 Env used for producing M- and T-SIV. This parental virus differs in nine amino acids, making it more resistant to antibody neutralization [24]. Using four different sera, we observed some inhibition of HIV (Fig 3C). Intriguingly, M-SIV was neutralized more efficiently than T-SIV. These effects were concentration-dependent (Fig 3D) and statistically significant (Fig 3E, paired t-test, p = 0.013). In light of these findings, we conclude that differences originating from the virus-producing cell can influence the efficacy of CBA and antibody neutralization, and thus might have relevance for the development of biomedical interventions based on these scaffolds.

Ethics statement

The animal studies were conducted at the German Primate Center, which has the permission to breed and house non-human primates under license number 392001/7 granted by the veterinary office of the city Göttingen and conforming with x 11 of the German Animal Welfare act. Approval was obtained from the ethics committee of the Lower Saxony State Office for Consumer Protection and Food Safety with the project license 33.14-42502-04-11/026. All animals were bred, cared for by qualified staff and, housed at the German Primate Center adhering to the German Animal Welfare Act and complying with the European Union guidelines for the use of nonhuman primates in biomedical research.

Animal studies

In total, eight male and one female Indian-origin rhesus macaques (Macaca mulatta) were assigned to experimental groups based on their age (4 to 7 years). A maximum of 5–6 ml blood per kg bodyweight was drawn ex vivo from animals, which were anesthetized intramuscularly with 10 mg ketamine per kg body weight from the femoral vein employing the Vacutainer system (BD Biosciences). For virus challenges, animals were anesthetized intramuscularly with a mixture of 5 mg ketamine, 1 mg xylazin, and 0.01 mg atropine per kg body weight. Virus introduction occurred up to ten centimeters into the rectum using a catheter (Urotech). During the procedure and for the subsequent 30 minutes, the animals were maintained in a ventral position with an elevated pelvis. Monitoring for infection establishment by RT-PCR took place every two and three weeks post-challenge.

All data underpinning this study, which is not already included in the manuscript, are publicly available at Dryad (https://datadryad.org/stash) with the identifier doi:10.5061/dryad.hmgqnk9rm [48].

Plasmids

The plasmids encoding HIV-1 NL4-3 [27], SIVmac239/316 Env [22], the HIV-1 NL4-3-derived vector pNL4-3.Luc.R-E- [49], and the vesicular stomatitis virus glycoprotein [50] have been previously described.

Cell culture

293T and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech) supplemented with 10% fetal bovine serum (FBS, Biochrome), 100 U/ml penicillin, and 100 μg/ml streptomycin (P/S; PAN-Biotech). The suspension cell lines (C8166, CEMx174 R5, Raji, Raji DC-SIGN/DC-SIGNR/Langerin) were cultured in RPMI 1640 supplemented with L-glutamine (PAN-Biotech), 10% FBS, and P/S. For the isolation of rhesus macaque primary CD4⁺ T cells and macrophages, PBMCs were isolated from whole blood using ficoll (Biochrom) density gradient centrifugation. CD4⁺ T cells were purified by negative depletion using magnetic microbeads (Miltenyi Biotech) following the manufacturer’s protocol and cultured in RPMI 1640 supplemented with 20% FBS, P/S and 10 μg/ml concanavalin A (Sigma-Aldrich) for 24 h at a density of 2 x 10⁶ cells/ml. Following that, CD4⁺ T cells were cultured in RPMI 1640 supplemented with 20% FBS, P/S and 100 U/ml recombinant human interleukin-2 (IL-2). For the generation of macrophages, monocytes were purified from PBMCs by positive selection for CD14⁺ cells with magnetic microbeads (Miltenyi Biotech) following the manufacturer’s protocol. For differentiation into macrophages, monocytes were seeded at a density of 3 x 10⁵ cells/ml in RPMI 1640 medium supplemented with 20% FBS, 10% human AB serum (Sigma-Aldrich), and 10 ng/ml recombinant human macrophage colony stimulating factor (Peprotech). The medium was replenished after 2 d and the cells differentiated for a total of 5 d. Subsequently, macrophages were cultured in RPMI 1640 supplemented with 20% FBS. All cells were grown in a humidified atmosphere at 37°C with 5% CO₂.

Flow cytometry

For flow cytometric analysis of marker expression, 50,000 to 500,000 PBMCs and the above mentioned cell subsets were stained for 30 min at room temperature (RT) with mixtures of monoclonal antibodies (mAb) reactive against CD3 (SP34-2, Alexa Fluor 700), CD4 (L200, V450), CD11b (ICRF44, PE), CD16 (3G8, FITC) and CD20 (L27, PE-Cy7) all from BD Biosciences, as well as CD8 (3B5, Pacific Orange, Invitrogen), and CD14 (RMO52, ECD, Beckman Coulter) diluted in staining buffer (phosphate-buffered saline with 5% FBS). Subsequently, cells were washed with staining buffer, and fixed with 4% paraformaldehyde solution for seven minutes. After an additional washing step, the cells were analyzed using a LSRII cytometer (BD Biosciences) equipped with three lasers. Compensation was calculated by FACS DIVA software 6.1.3 using appropriate single antibody labeled compensation beads from SpheroTech. Data analysis was performed using FlowJo software v9.6 (Treestar).

Production of viruses and pseudotyped viruses

To generate virus stocks of HIV-1 NL4-3 and SIVmac239/316 Env, 293T cells were seeded into T25-cell culture flasks and transfected with 12 μg of plasmids encoding proviral DNA using calcium phosphate. For pseudotype production, 293T cells were cotransfected with plasmids encoding pNL4-3.Luc.R-E- and VSV-G. The culture medium was exchanged at 6–7 h post transfection, and the cellular supernatant was harvested at 72 h post transfection. The supernatants were clarified from debris by centrifugation (5 min, 3488 x g, RT) filtered through a 0.45 μm filter, aliquoted and stored at -80°C.

Amplification of SIV in T cells and macrophages

To produce SIVmac239/316 Env in CD4⁺ T cells, the concanavalin A stimulated cells were infected with SIVmac239/316 Env generated in 293T cells at a multiplicity of infection (MOI) of 0.1 in RPMI 1640 medium supplemented with 20% FCS and P/S at RT under occasional shaking. Subsequently, the cells were grown in medium supplemented with IL-2 and incubated for 48 h. The cells were then washed twice with 5 ml of culture medium, transferred to a new cell culture flask, and cultured for 2 weeks. Every 2–3 d, the cells were pelleted, the supernatant was harvested and the cells were dissolved in fresh media at a density of 2 x 10⁶ cells/ml. The supernatants were processed as described above for pseudotypes and viral capsid protein concentration determined by a p27-antigen capture enzyme linked immunosorbent assay (ABL), following the manufacturer’s instructions. The p27-antigen-positive supernatants from CD4⁺ T cell cultures obtained from 9 donor animals were pooled to create the stock of CD4⁺ T cell-derived SIVmac239/316 Env, referred to as T-SIV throughout the manuscript. To generate SIVmac239/316 Env in macrophages (M-SIV), the same procedure as described above was followed, except that washing and harvesting of the cells were conducted without detaching the cells from the cell culture flask, and no IL-2 was added to the cell culture medium. The M-SIV virus stock was derived from infected cultures established from eight donor animals. To confirm the absence of mutations in env introduced during virus replication, we isolated RNA from M-SIV and T-SIV using the High Pure Viral RNA Kit (Roche), converted it to cDNA with the Cloned AMV First-Strand cDNA Synthesis Kit (Invitrogen), and then sequenced it after PCR amplification. The viral stocks were further characterized for p27-capsid content, as described above, and for infectious units/ml by titration on C8166 cells as described before [51].

Infectivity assays

For the determination of virus stock infectivity, TZM-bl cells, the standard indicator cell line for neutralization assays using clinical samples [34], were utilized. The cells were seeded at a density of 10,000 cells per well in a 96-well cell culture plate and allowed to adhere for 2 hours prior to infection. Infection was carried out using p27-capsid protein or MOI normalized SIVmac239/316 Env in a total volume of 100 μl. After 2 h of spin-oculation (870 x g, RT) [52], the infection was allowed to continue for 3–4 h at 37°C. Thereafter, the infection medium was replaced by 200 μl of fresh culture medium and the cells were cultured for 72 h. Subsequently, the cells were lysed and beta-galactosidase activity in lysates was detected using a commercially available kit (Applied Biosystem) following the manufacturer’s protocol. For lectin inhibition assays, infectivity normalized M-SIV, T-SIV, HIV-1 NL4-3 and env-defective NL4-3 pseudotyped with VSV-G were preincubated with PBS or the lectins UEA (Eylabs), GNA (Sigma), or CV-N [38] for 15 min at 37°C. Subsequently, the lectin-virus mix was added to TZM-bl cells for infection, and the infection efficiency was determined as described earlier. To assess antibody-mediated neutralization, a similar experimental procedure as the lectin inhibition assay was carried out, except that sera obtained from SIVmac239-infected rhesus macaques were used instead of lectins. Before use, the sera were heat-inactivated for 30 minutes at 56°C.

Transmission assays

To model viral transmission we followed a previously published protocol [31]. Briefly, 30,000 Raji, Raji DC-SIGN, Raji DC-SIGNR, or Raji Langerin cells were preincubated for 2–3 h at 37°C with virus adjusted to ensure equivalent infectivity on the CEMx174 R5 target cell line. Subsequently, the cells underwent two washes with 5 ml PBS each (270 x g, 5 min) to eliminate unbound virus. Following this, the cells were co-cultured with 30,000 CEMx174 R5 target cells in 100 μl of RPMI-1640 medium in a 96-well cell culture plates. After two days, 50 μl of the medium was replaced with fresh media. One day thereafter, the cells were lysed, and luciferase activity was quantified utilizing a commercially available assay kit (Promega). In addition, all cell lines were infected directly without subsequent removal of unbound viruses to control the uniform infectivity of viruses and background signals of transmitter cell lines.

Western blot

For the analysis of viral particle content, the virus was concentrated through a 20% sucrose cushion in TNE buffer (0.01 M Tris-HCl pH 7.4, 0.15 M NaCl and 2 mM EDTA in ddH₂O) using centrifugation. The proteins from the pelleted virions were then separated using SDS-PAGE and subjected to western blot analysis. SIV gp120 was detected using the mouse monoclonal gp120-specific antibody DA6 [53] at a dilution of 1:2,000, while the mouse monoclonal p27-specific antibody 55-2F12 [54] was employed at a dilution of 1:100 for the detection of p27-capsid protein. As a secondary antibody, a horseradish peroxidase-labeled antibody of appropriate species specificity from Dianova was employed at a dilution of 1:5,000. In order to examine the glycosylation of gp120, the concentrated virus was treated with either Endo H or PNGase F from New England Biolabs, for 30 min prior to SDS-PAGE. Signal intensities of western blot bands were quantified using the software ImageJ [55].

Glycoprofiling by xCGE-LIF

To investigate the N-glycosylation of gp120 from M- and T-SIV, sample preparation and analysis were performed as described before [56]. Briefly, the virions were concentrated by ultra-centrifugation through a sucrose cushion and the viral proteins were separated by SDS-PAGE. The gp120 protein bands were excised from the Coomassie Blue-stained SDS-polyacrylamide gels, destained, reduced, and alkylated. The attached N-glycans were then released by in-gel incubation with PNGase F, and the released N-glycans were extracted with water. Next, the N-glycans were labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), and any excess label was removed using hydrophilic interaction solid phase extraction. The fluorescently labeled N-glycans were separated and analyzed by xCGE-LIF. The glyXtoolCE software (glyXera) was utilized to process the data generated by xCGE-LIF, including the normalization of migration times to an internal standard. This resulted in the creation of N-glycan "fingerprints" where the signal intensity in relative fluorescence units (RFU) was plotted on the y-axis against the aligned migration time in aligned migration time units (MTU") on the x-axis. The high reproducibility of aligned migration times allowed for the comparison of N-glycan "fingerprints" between different samples. To elucidate the N-glycan structures and annotate the peaks, an in-house N-glycan database was used. For quantitative comparison, the relative peak height, which represents the ratio of the peak height to the total height of all peaks, was calculated for each peak and sample.

LC-MS/MS and automated MS data analysis

After the in-gel release of N-glycans by PNGase F treatment proteins were digested with trypsin according to the method outlined by Shevchenko et al. [57]. The procedure involved reducing the proteins with 10 mM DTT (Sigma-Aldrich), followed by carbamidomethylation with 100 mM iodocetamide (Sigma-Aldrich), and subsequent digestion with sequencing-grade trypsin (Promega). To extract the resulting peptides, acetonitrile was used, and the samples were then dried in a vacuum centrifuge before being dissolved in a solution containing 2% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid (Sigma-Aldrich) for subsequent LC-MS/MS analysis. The analysis was performed using a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) coupled online to a nano-flow ultra-high-pressure liquid chromatography system (RSLC, Thermo Fisher Scientific). Reverse-phase chromatography and mass spectrometry was carried out as described previously [58]. For data analysis, the MaxQuant proteomics software suite version 1.2.2.5 [59] was utilized, and peak lists were searched against the SIVmac239/316 Env sequence using the Andromeda search engine version 1.1.0.36 [60].

Software

Graphs and statistics were conducted using the GraphPad Prism 9 (Dotmatics) software unless stated otherwise. The text of this manuscript was subjected to rephrasing using ChatGPT (OpenAI) to enhance its linguistic quality.