Kennedy epitope (KE)-dependent retrograde transport of efficiently cleaved HIV-1 envelopes (Envs) and its effect on Env cell surface expression and viral particle formation

doi:10.21203/rs.3.rs-2245790/v1

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Kennedy epitope (KE)-dependent retrograde transport of efficiently cleaved HIV-1 envelopes (Envs) and its effect on Env cell surface expression and viral particle formation

https://doi.org/10.21203/rs.3.rs-2245790/v1

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published 04 Oct, 2023

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Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 − 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 − 2.J41 and JRCSF Envs increases but particle formation decreases for 4 − 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes.

HIV-1

envelope

efficiently cleaved

C-terminal tail

Kennedy epitope

retrograde transport

A major goal of a HIV-1 vaccine is to elicit broadly neutralizing antibodies (bNAbs) by activating the humoral immune response using HIV-1 Env-based immunogens. The Env glycoprotein is heavily glycosylated (about 50% of its molecular mass is composed of glycans) which occludes conserved regions in the protein that can be targeted by neutralizing antibodies. Nonetheless, there are specific epitopes in Env including glycan and conformation-dependent ones which can be targeted by bNAbs (1–6). Several of these bNAbs when delivered through passive immunization protect from HIV-1 infection by neutralizing viruses, preventing infection and by rapidly clearing viruses and infected cells, in animal models (7), suggesting that eliciting these bNAbs through vaccination will be protective. Currently, the paradigm for developing such a HIV-1 vaccine is to generate Env-based immunogens that can elicit one or more of these bNAbs upon immunization (2, 5, 8, 9).

The HIV-1 Env glycoprotein in its functional, native form is a trimer of a heterodimer of the soluble gp120 and transmembrane gp41 subunits that are formed by furin-mediated cleavage of the precursor gp160 polypeptide and the subunits are held together by non-covalent association (10). We and others have shown that efficient cleavage of the gp160 precursor polypeptide into constituent subunits during Env maturation leads to selective binding of Env to bNAbs as opposed to non-NAbs (non-neutralizing antibodies), a property that is necessary for any Env-based immunogen (11–16). Uncleaved Envs, on the other hand, mostly bind to both neutralizing and non-neutralizing antibodies (11–15). Using a six step strategy (17), we have identified the Envs A5 and BG505 (African clade A), JRCSF (American clade B), 4 − 2.J41 (Indian clade C) and LT5.J4b12C (Indian clade B/C) as efficiently cleaved on the cell surface and showing excellent antigenic properties (13–16). Thus, these Envs can be used to deliver immunogens by different methods viz virus-like particles, viral vectors or plasmid DNAs as well as converted into soluble proteins (18).

The C-terminal tail (CT) of Env contains several conserved domains that regulate different Env functions (19–23). We have previously reported that deletion of the CT of naturally occurring, efficiently cleaved HIV-1 Envs have differential effect on their ectodomain (ET) antigenicity and conformation on the cell surface (24). While JRFL (clade B) and A5 (clade A) show no detrimental effect of CT-deletion on ET integrity, JRCSF (clade B) and 4 − 2.J41 (clade C) show defective ET conformation and antigenicity (24). In the latter, the wild-type conformation and antigenicity can be restored by restoring a conserved hydrophilic domain (CHD), also called Kennedy epitope (KE), in the CT (24), suggesting that this domain plays a critical role in cell surface ET conformation and antigenicity for a subset of efficiently cleaved Envs (24). This observation with CT-deleted 4 − 2.J41 and JRCSF Envs is largely limited to the cell surface protein as CT deletion for these Envs has only modest effects at the level of pseudovirus neutralization suggesting that native or near-native Envs are specifically incorporated into virions in the presence of one or more viral protein (24). However, the mechanism by which the CHD/KE differentially regulates the ectodomain conformation/integrity of efficiently cleaved Envs is not known.

The CT of HIV-1 Env plays a central role in protein sorting through interactions with various components of the cellular trafficking machinery (25). The conserved motifs in CT interact with AP-1 and AP-2 adapter complexes for intra-cellular trafficking in a complex mechanism, which is yet to be clearly understood (26, 27). Furthermore, the regulated intracellular trafficking of Env in ER-Golgi network plays an important role in formation of the mature gp120-gp41 trimer complex in the secretory pathway to the plasma membrane (22, 23), which may be critical for native-like ectodomain conformation. Additionally, CT-mediated retromer-dependent transport of Env from endosomes to golgi plays a role in Env incorporation into viral particles (28). HIV-1 Env is localized to the Golgi complex and traverses to the plasma membrane where it is endocytosed (29) and undergoes retrograde transport from endosome to Golgi complex through interaction of the CT with different adapter proteins (29–31). As the CTs of Envs play a key role in their trafficking within cells, we looked at whether cellular transport pathways of efficiently cleaved Envs play a role in determining their differential CHD/KE-dependent properties.

Here, we report that retrograde transport plays an important role in the differential properties of the efficiently cleaved Envs that we identified. Those Envs which show a C-terminal tail, CHD/KE domain-mediated effect on cell surface ectodomain (ET) antigenic integrity like 4 − 2.J41 and JRCSF behave differently from those Envs like JRFL where deletion of the CT does not have an effect on ET integrity. We show a defect in 4 − 2.J41 and JRCSF Envs but not JRFL Env in their ability to undergo retrograde transport from the early to late endosomes that is dependent on the CHD/KE domain in their CT. Using the retrograde transport inhibitor Retro 2 (32–34), we show a differential effect on cell surface expression and pseudoviral particle formation (measure of transport, assembly and release of virus) that segregates the Envs according to the effect of their CT on cell surface ET integrity. The implications of these findings are discussed.

2.1 Cell lines, reagents and antibodies

293T cells were obtained from the American Type Culture Collection (ATCC). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) that was supplemented with 10% heat inactivated-fetal bovine serum (HI-FBS) (HiMedia, USA), 20 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Retro 2 was obtained from Sigma-Aldrich (Cat# SML-1085). The anti-gp120 (clades B, C) rabbit polyclonal antibodies were obtained from ABLinc, USA. EEA-1 (early endosomal autoantigen 1) antibody (Cat# NBP1-30914) and CD63 (CD63 antigen) antibody (Cat # NB100-77913) were obtained from Novus Biologicals.

2.2 Plasmids and mutagenesis

The 4 − 2.J41 Env expressing clone was obtained from National AIDS Research Institute (NARI), Pune, India; JRFL and JRCSF expressing clones from the Neutralizing Antibody Center (NAC), The Scripps Research Institute, La Jolla, USA. CT deleted constructs were generated by introducing stop codons into the cytoplasmic tail at the amino acid (aa) positions mentioned using PCR-mediated mutagenesis as described (24).

2.3 Endosomal fraction isolation, lysate preparation, silver staining, western blot analysis

Early and late endosomal (EE and LE) fractions were isolated by the method previously described by de Araujo et al (35, 36). Briefly, 293T cells were transfected with respective plasmids, harvested and incubated in HBA (3 mM Imidazole pH 7.4, 1 mM EDTA) on ice for 20 min. Following incubation, equal volume of buffer HBB (250 mM sucrose, 3 mM Imidazole pH 7.4, 1 mM EDTA) was added and the suspension passed through a 24-gauge syringe needle 10 times. Supernatant was diluted with 0.7 vol HBA, cycloheximide added to a final concentration of 0.05 mM and clarified twice by centrifugation to get PNS (post-nuclear supernatant). The PNS was used to isolate EE and LE fractions by a sucrose step gradient as described (35, 36) and the endosomal fractions further pelleted by centrifugation after dilution in HBA. Lysates were prepared from pellets using lysis buffer [PBS (phosphate buffered saline), 1% Triton-X, protease inhibitors], centrifuged, protein concentration determined by BCA (Bicinchoninic Acid) protein assay kit (Pierce/Thermo Fisher Scientific) and equal amount of protein analyzed by western blot analysis using anti-Env polyclonal antibodies. Bands on western blots were quantified using the ImageJ software and fold change determined from ratio of band intensity in EE versus LE lanes. In order to determine equal protein loading in EE and LE lanes, SDS-PAGE of equal amount of protein from early and late endosomal fractions followed by silver staining was carried out using the Pierce Silver Stain kit (ThermoFisher Scientific). Western blot analysis of isolated early and late endosomal fractions were carried out by using anti-EEA1 and anti-CD63 antibodies as probes for early and late endosomes, respectively.

2.4 FACS-based cell surface antibody binding assay

FACS-based cell surface antibody binding assay was carried out with 293T cells transfected with plasmids encoding 4 − 2.J41, JRFCSF and JRFL Envs (15 µg) and pctat plasmid (ratio of Env:Tat = 20:1) in 100 mm plates as described (17). Following transfection, the next day cells were incubated overnight in the absence and presence of 25 µM Retro 2. Cells were harvested 36–48 hours post-transfection, counted using a hemocytometer, plated into 96 well U-bottom plates and stained with the neutralizing antibody PGT121 and non-neutralizing antibodies F105, 17b, 19b in duplicate as described (17). R-Phycoerythrin-conjugated AffinityPure F(ab’)₂ Fragment Goat Anti-Human IgG (Cat# 109-116-097; Jackson ImmunoResearch Laboratories Inc.) secondary antibody was used (17) and stained cells analyzed by FACS Canto II analyzer (BD Biosciences) to determine MFI (mean fluorescent intensity). Data were analyzed by FlowJo software v10.

2.5 ELISA (Enzyme-linked Immune Sorbent Assay) for detection of HIV-1 p24

293T cells plated one day before transfection in T25 flasks were transfected with the HIV-1 backbone vector SG3ΔEnv and Env expressing plasmids for 4 − 2.J41 and JRCSF. The next day media was changed and cells incubated in the absence or presence of 25 µM Retro 2 overnight. The following day supernatant and cells were harvested. Supernatant was clarified by centrifugation and pelleted cells were lysed in buffer containing PBS + 1% Triton-X and protease inhibitors, and lysates were clarified by centrifugation. The p24 in supernatant and lysate were measured by ELISA, after appropriate dilution, using HIV-1 p24 antigen capture assay (Advanced Bioscience Laboratories, ABLinc). All the components used in this assay including microelisa plates were kept in room temperature. 25 µl of disruption buffer was added to each well of microELISA plates used in this assay. HIV-1 p24 standard in complete tissue culture media with different dilution (100, 50, 25, 12.5, 6.3, and 3.1 pg/ml) was used. 100 µl of each diluted HIV-1 p24 standard was added to microELISA plate well containing disruption buffer in duplicate. In case of negative controls, 100 µl of complete tissue culture media (containing 10% FBS) was added to 4 wells containing disruption buffer. 100 µl of prepared test samples in duplicate were used in microELISA plate wells containing disruption buffer. The wells in the plate were mixed gently, covered with plate sealer and incubated at 37 ⁰C for 1 h. MicroELISA plate was washed with 300 µl of wash buffer 4 times followed by addition of 100 µl of conjugate solution to each well and the plate incubated for 1 h at 37 ⁰C. The microELISA plate was washed as earlier. 100 µl of peroxidase substrate was added to each well and incubated for 30 min at room temperature. After color formation, the reaction was stopped by using 100 µl of stop solution containing 2N sulphuric acid. The plate absorbance was read at 450 nm in a microELISA plate reader within 20 min.

3.1 Retrograde transport is affected in 4 − 2.J41/JRCSF Envs, whose CHD/KE domain regulate ectodomain integrity, upon CT-deletion, but not in JRFL Env

We investigated biochemically the distribution of efficiently cleaved Envs in early and late endosomal fractions, key components of the retrograde transport pathway. We used 293T cells, which cell line and its derivatives are commonly used to study and produce HIV-1 Envs, e.g. cell surface antibody binding studies, pseudovirus production, virus-like particle production and protein immunogen production. We kept the transfection conditions similar to what we use for determining cell surface antibody binding. We first looked at the efficiently cleaved clade C Env, 4 − 2.J41 and clade B Env, JRCSF; deletion of the C-terminal tail of these Envs adversely affects their ectodomain integrity by exposing non-neutralizing epitopes (13, 24). As a control, we looked at the distribution of the clade B Env, JRFL whose ectodomain integrity is not altered upon deletion of the CT (24). A representative example of the constructs we used for this study is shown in Fig. 1. We isolated early endosomal (EE) and late endosomal (LE) fractions from 4 − 2.J41_WT, JRCSF, JRFL and 4 − 2.J41∆CT₇₁₁, JRCSFΔCT, JRFL∆CT transfected 293T cells by the method of de Araujo et al (35, 36), and then carried out western blot analysis of their lysates (Fig. 2). We compared the wild-type and ∆CT Envs side-by-side.

First, we loaded equal amount of protein extracted from the early and late endosomal fractions in SDS-PAGE and verified similar level of protein extraction in each compartment by silver staining (Fig. 2A). In silver stains of isolated early and late endosomal fractions several of the bands were enriched in one or the other compartment (shown by arrows in Fig. 2A). To further confirm that our endosomal preparations were enriched in early and late endosomes we carried out western blot analysis of the isolated fractions using antibodies specific for early endosome marker EEA-1 and late endosome marker CD63 and found that the isolated early endosomal preparations were enriched for EEA-1 and the late endosomal preparations were enriched for CD63 (Fig. 2B). In western blots, the Envs migrate as two bands with the slower migrating band likely to be gp160 and its CT truncated versions (marked with * in Fig. 2, 3). JRFL and JRFL∆CT Env controls showed similar distribution in both EE and LE compartments (Fig. 2C) while in 4 − 2.J41 and JRCSF Envs there was much more Env protein in EE than in LE fractions (Fig. 2D, 2E). This may be due to lysosomal degradation of these Envs. The distribution of 4 − 2.J41∆CT₇₁₁ Env showed a higher proportion of Env protein in EE versus LE fractions as compared to the wild-type protein (Fig. 2D) suggesting a defect in transport from EE to LE compartments and accumulation of CT-deleted Env protein in EE fraction. In addition, lysates of EE and LE fractions of JRCSF and JRCSF∆CT transfected 293T cells showed higher proportion of Env protein in EE versus LE compartments in JRCSF∆CT as compared to the wild-type protein (Fig. 2E) suggesting that upon CT deletion retrograde transport from EE to LE of JRCSF∆CT is also defective leading to accumulation of Env protein in EE fraction. Taken together, these results suggest that deletion of the CT of JRCSF and 4 − 2.J41, but not JRFL, leads to a defect in retrograde transport from the early to late endosomes, which correlate with the effect of their CT on cell surface ET antigenicity/conformation.

3.2 Retrograde transport is restored in 4 − 2.J41∆CT₇₅₃ and JRCSF∆CT₇₅₉ Envs similar to wild-type Envs

We next evaluated the distribution of 4 − 2.J41∆CT₇₅₃ and JRCSF∆CT₇₅₉ Envs in endosomal compartments biochemically. In these mutant Envs, where the CHD/KE is restored, the native-like cell surface antigenicity/conformation properties are also restored (24). We investigated whether retrograde transport of 4 − 2.J41∆CT₇₅₃ Env from EE to LE compartments is restored similar to wild-type levels by carrying out western blot analysis of isolated EE and LE fraction lysates from 4 − 2.J41_WT, 4 − 2.J41∆CT₇₁₁ and 4 − 2.J41∆CT₇₅₃ Env transfected 293T cells (Fig. 3A). Densitometric scanning was used to quantitate the amount of Env protein in both endosomal compartments. The proportion of Env protein in EE versus LE fractions is restored in 4 − 2.J41∆CT₇₅₃ Env which contains the CHD/KE domain (ratio approximately 6x in EE to that of LE fraction) similar to the wild-type protein (ratio approximately 5x in EE to that of LE fraction) in contrast to 4 − 2.J41∆CT₇₁₁ Env (ratio approximately 8x in EE to that of LE fraction) (Fig. 3A). In agreement with this finding, we also observed in western blot analysis of isolated EE and LE fraction lysates obtained from transfected JRCSF/JRCSF∆CT/JRCSF∆CT₇₅₉ 293T cells that defective distribution of JRCSF∆CT (ratio approximately 7x in EE to that of LE fraction) in the endosomal compartments, as compared to the wild-type protein (ratio approximately 4x in EE to that of LE fraction), is restored in JRCSF∆CT₇₅₉ Env which contains the CHD/KE domain (Fig. 3B). In fact, we observed more JRCSF∆CT₇₅₉ Env in LE compartment as compared to the EE compartment (ratio approximately 0.5x in EE to that of LE fraction) (Fig. 3B). Taken together these studies show that in transfected 293T cells those Envs, whose CT determine their ectodomain integrity/conformation, restoration of the CHD/KE domain in their CT restores the relative distribution of Env protein in endosomal compartments just as they restore cell surface antigenicity/conformation.

3.3 Retrograde transport inhibitor Retro 2 has differential effect on cell surface expression of efficiently cleaved Envs

In order to directly interrogate whether retrograde transport differentially affects efficiently cleaved Envs we used the retrograde transport inhibitor Retro 2 which blocks transport from endosomes to golgi (32–34) and studied different properties of Envs in transfected 293T cells. We first looked at cell surface antigenicity/conformation of Envs in 293T cells transfected with 4 − 2.J41, JRCSF and JRFL in the absence and presence of 25 µM Retro 2 and carried out FACS-based cell surface antibody binding experiment with the V3-directed broadly neutralizing antibody PGT121 and the non-neutralizing antibodies F105 [CD4 (cluster of differentiation 4)-binding site directed], 17b [CD4i (CD4-induced) directed] and 19b [V3 (variable region 3) region] (Fig. 4). Both 4 − 2.J41 and JRCSF Envs showed an increase in binding to PGT121 upon Retro 2 treatment (Fig. 4, bottom panels). Binding to PGT121 by 4 − 2.J41 Env increased from 38.1–45.8% upon Retro 2 treatment whereas for JRCSF it increased from 33.3–39.2% upon Retro 2 treatment (Fig. 4, bottom panels). Binding to the non-neutralizing antibodies did not change much (Fig. 4, bottom panels). JRFL Env on the other hand showed a small decrease in binding to PGT121 on the cell surface upon Retro 2 treatment (Fig. 4, top panel). Taken together these studies demonstrate that the retrograde transport inhibitor Retro 2 has an effect on some efficiently cleaved Envs. In the case of Envs like 4 − 2.J41 and JRCSF, which show a CHD/KE domain-mediated, CT-truncation dependent effect on cell surface antigenicity/conformation, Retro 2 treatment increases cell surface expression whereas for JRFL, whose CT truncation does not have a detrimental effect on cell surface antigenicity/conformation, cell surface expression did not change much. Binding to non-neutralizing antibodies did not change much for any of the studied Envs.

3.4 Retrograde transport inhibitor Retro 2 has differential effect on formation of pseudoviral particles containing efficiently cleaved Envs

As retrograde transport plays a role in Env incorporation into viral particles mediated by its C-terminal tail we studied the effect of the retrograde transport inhibitor Retro 2 (32–34) in viral particle (pseudoviruses) formation with the efficiently cleaved Envs 4 − 2.J41, JRCSF and JRFL by p24 ELISA of both intracellular p24 and virion associated p24 and then determined the ratio of virion associated to intracellular p24 which is an indirect measure of assembly, release leading to viral particle formation (37, 38). As described in Materials and Methods, 293T cells were transfected with backbone vector SG3ΔEnv and Envs and then left untreated or treated with 25 µM Retro 2 and the p24 in cell lysate and supernatant determined by ELISA. Co-transfection of backbone vector and Env into 293T cells typically leads to Env incorporation and pseudoviral particle formation. Retro 2 treatment did not alter the level of expression of the Envs as 4 − 2.J41, JRCSF and JRFL Envs were expressed to a similar level in the absence and presence of Retro 2 as shown in western blots (Fig. 5A-C). The ratio of virion associated versus intracellular p24 was determined from total p24 in cell lysate and supernatant as shown in Fig. 5D-F. Both 4 − 2.J41 and JRCSF Envs containing pseudoviral particles showed a decrease in ratio, JRCSF more prominently than 4 − 2.J41, while JRFL had an opposite effect and showed an increase in ratio of virion associated to intracellular p24 (Fig. 5D-F) suggesting that Retro 2 has differential effect on assembly, release leading to viral particle formation of the efficiently cleaved Envs that correlates with the effect of CT truncation on their cell surface antigenicity/conformation and defect in retrograde transport from early to late endosomes.

In order to generate mature, efficiently cleaved, functional Envs, the HIV-1 Env, following synthesis in the ER, has to traverse through the Golgi/trans-Golgi network to the plasma membrane during which it undergoes proteolytic processing as well as post-translational modifications (25, 39). The endosomal system is a complex network that sorts cargoes to different subcellular compartments like export from endosomes through retrograde transport pathways, recycles to the plasma membrane and transports them to the lysosome for degradation (40). HIV-1 Env undergoes retrograde transport during intracellular trafficking of Env in a retromer-dependent manner (28) and disrupting retromer integrity affects Env incorporation into nascent virions (28). This led us to study the transport/trafficking of the efficiently cleaved Envs 4 − 2.J41, JRCSF and JRFL Envs to understand whether they behave differently which may give insights into their differential properties.

We isolated EE (early endosomal) and LE (late endosomal) fractions from transfected 293T cells and demonstrate a defect in transport from the EE to LE compartments by western blot, leading to accumulation of the protein in early endosomal fractions, in case of 4 − 2.J41∆CT and JRCSFΔCT Env proteins but not in JRFL∆CT Env protein. These results suggest that retrograde transport of naturally occurring, efficiently cleaved Envs depend on their CT. Thus, retrograde transport of 4 − 2.J41∆CT and JRCSFΔCT Env proteins, whose CT deletion affects their ET integrity, are also affected by CT truncation. However, this is not the case with JRFL∆CT Env whose retrograde transport remains unaltered upon CT deletion and whose ET antigenicity/conformation also remains unaffected by CT truncation. It is to be noted here that we observed less Env in LE fractions as compared to EE fractions for both 4 − 2.J41 and JRCSF Envs but not JRFL Env which may be due to 4 − 2.J41 and JRCSF Envs entering the lysosomal degradation pathway.

We also evaluated the role of the highly conserved hydrophilic domain (CHD) or Kennedy epitope (KE) in the CT of Envs (22, 41), which restores cell surface ET conformation/antigenicity of 4 − 2.J41 and JRCSF Envs (24), in retrograde transport and found that indeed restoration of the CHD/KE-containing region in 4 − 2.J41ΔCT₇₅₃ and JRCSFΔCT₇₅₉ Envs restored the wild-type retrograde transport pattern in early and late endosomal fractions. Based on our data we hypothesize that viruses containing different efficiently cleaved Envs may be using different pathways for viral assembly and release (Fig. 6). While viruses containing Envs like 4 − 2.J41 and JRCSF may be using the endosomal pathway for assembly and release, JRFL containing viruses are likely using the plasma membrane. This differential effect is dependent on the conserved hydrophilic domain (CHD) or Kennedy epitope (KE) of 4 − 2.J41 and JRCSF Envs which may be interacting with host cellular proteins involved in retrograde transport from the EE to LE compartments, a process necessary for proper assembly and release of viruses containing these Envs. Envs like JRFL which do not show a detrimental effect of CT deletion on ET integrity may have a different mechanism and/or different interaction partners and viruses containing this Env is assembled and released from the plasma membrane. We have previously shown that the known major motifs in the C-terminal tail of 4 − 2.J41, JRFL and JRCSF are conserved (24) suggesting that some novel mechanism(s) must be involved in these differential effects. One possibility is that two alternative models have been proposed for the orientation of the CT, particularly the CHD/KE (41), which may lead to different interaction partners and mechanisms leading to different transport pathways.

To further corroborate our findings, we studied the effect of the retrograde transport inhibitor Retro 2 on cell surface expression and formation of viral particles containing these Envs. Upon treatment with the retrograde transport inhibitor Retro 2, cell surface expression of 4 − 2.J41 and JRCSF Envs increased suggesting diversion of these proteins towards the plasma membrane pathway instead of the retrograde transport pathway. Treatment with Retro 2 caused a decrease in pseudovirus particles containing 4 − 2.J41 and JRCSF Envs suggesting that these Envs may be using the retrograde transport pathway for assembly and release. Diversion of these Envs from the retrograde transport pathway causes a reduction in pseudoviral particle formation. JRFL Env, on the other hand, had a different effect. Although we did not observe a significant change in cell surface expression, we did observe an increase in pseudoviral particle formation which may be due to diversion of the protein towards the plasma membrane pathway, the pathway which leads to assembly and release of viruses containing Envs like JRFL. Our results also suggest that these efficiently cleaved Envs have different mechanisms and pathways of transport and assembly of Envs leading to formation of viral particles. It is to be noted here that different HIV-1 isolates show different tropism for CD4 T cells and macrophages (42). Assembly and release of HIV-1 virions in T cells and macrophages seem to differ (42). While in T cells it occurs at the plasma membrane, in macrophages viruses assemble in late endosomes/multivesicular bodies (MVB) and are released either by late endosome fusion with plasma membrane or bud into the lumen of endocytic organelles which are then released as exosomes via fusion of MVB with plasma membrane (42).

Our studies are limited to these Envs as the number of efficiently cleaved Envs that have been identified is very limited. In future, these studies can be expanded to more efficiently cleaved Envs when they are identified. We have used a 293T cell transfection system for studying the properties of these efficiently cleaved Envs as this cell line and its derivatives are commonly used for cell surface antibody binding studies, pseudovirus production, virus-like particle production and protein immunogen production. While overexpression systems have their own limitations, they can still provide important initial insights into mechanisms and pathways. In future, it will be important to study the mechanisms and pathways that regulate different properties of these efficiently cleaved Envs, that we demonstrate here, in other cell lines including those that are physiologically more relevant to HIV-1 e.g. T cell lines.

Competing interests: The authors declare that they have no competing interests.

Funding: This work was supported primarily by IAVI intramural research, as well as by the Department of Biotechnology (DBT), Govt. of India intramural research program and its generous donors. IAVI’s work is made possible by generous support from many donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). The full list of IAVI donors is available at www.iavi.org. This study is made possible by the generous support of the American people through USAID. The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government.

Acknowledgements: We thank Dr. Bimal Chakrabarti, ex-Program Director, THSTI-IAVI HIV Vaccine Design Program, Translational Health Science and Technology Institute, India for helpful discussions.

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published 04 Oct, 2023

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Kennedy epitope (KE)-dependent retrograde transport of efficiently cleaved HIV-1 envelopes (Envs) and its effect on Env cell surface expression and viral particle formation

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell lines, reagents and antibodies

2.2 Plasmids and mutagenesis

2.3 Endosomal fraction isolation, lysate preparation, silver staining, western blot analysis

2.4 FACS-based cell surface antibody binding assay

2.5 ELISA (Enzyme-linked Immune Sorbent Assay) for detection of HIV-1 p24

3. Results

3.1 Retrograde transport is affected in 4 − 2.J41/JRCSF Envs, whose CHD/KE domain regulate ectodomain integrity, upon CT-deletion, but not in JRFL Env

3.2 Retrograde transport is restored in 4 − 2.J41∆CT₇₅₃ and JRCSF∆CT₇₅₉ Envs similar to wild-type Envs

3.3 Retrograde transport inhibitor Retro 2 has differential effect on cell surface expression of efficiently cleaved Envs

3.4 Retrograde transport inhibitor Retro 2 has differential effect on formation of pseudoviral particles containing efficiently cleaved Envs

4. Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Kennedy epitope (KE)-dependent retrograde transport of efficiently cleaved HIV-1 envelopes (Envs) and its effect on Env cell surface expression and viral particle formation

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell lines, reagents and antibodies

2.2 Plasmids and mutagenesis

2.3 Endosomal fraction isolation, lysate preparation, silver staining, western blot analysis

2.4 FACS-based cell surface antibody binding assay

2.5 ELISA (Enzyme-linked Immune Sorbent Assay) for detection of HIV-1 p24

3. Results

3.1 Retrograde transport is affected in 4 − 2.J41/JRCSF Envs, whose CHD/KE domain regulate ectodomain integrity, upon CT-deletion, but not in JRFL Env

3.2 Retrograde transport is restored in 4 − 2.J41∆CT753 and JRCSF∆CT759 Envs similar to wild-type Envs

3.3 Retrograde transport inhibitor Retro 2 has differential effect on cell surface expression of efficiently cleaved Envs

3.4 Retrograde transport inhibitor Retro 2 has differential effect on formation of pseudoviral particles containing efficiently cleaved Envs

4. Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

3.2 Retrograde transport is restored in 4 − 2.J41∆CT₇₅₃ and JRCSF∆CT₇₅₉ Envs similar to wild-type Envs