HIV-1 Gp120 Research Articles

Fostemsavir: a first-in-class HIV-1 attachment inhibitor.

Fostemsavir is a recently Food and Drug Administration-approved HIV-1 attachment inhibitor that binds to HIV-1 gp120 and prevents viral attachment to the cellular CD4 receptor. Here, we review the pharmacology, efficacy, tolerability, and resistance profile of fostemsavir. Fostemsavir is well tolerated and maintains virologic activity in individuals harboring multidrug-resistant HIV-1. In conjunction with optimal background therapy, a majority of heavily treatment-experienced clinical trial participants treated with fostemsavir achieved virologic suppression. The approval of fostemsavir represents an important advance for individuals harboring multidrug resistant HIV-1 due to its novel mechanism of action and lack of cross-resistance to other antiretrovirals. Further study will better define the role of resistance testing for fostemsavir and fostemsavir's potential role outside of salvage therapy in heavily treatment-experienced individuals.

Click chemistry and molecular modeling methods in computer-aided design and identification of potential HIV-1 inhibitors

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to perform the computer-aided design of potential HIV-1 inhibitors able to block the membrane- proximal external region (MPER) of HIV-1 gp41 that plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds that exhibit the high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Developing a secretory AcGFP1-based IRES expression system for efficient production of mammalian recombinant proteins

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.

Blocking T-cell egress with FTY720 extends DNA vaccine expression but reduces immunogenicity.

Optimal immunogenicity from nucleic acid vaccines requires a balance of antigen expression that effectively engages the host immune system without generating a cellular response that rapidly destroys cells producing the antigen and thereby limiting vaccine antigen expression. We investigated the role of the cellular response on the expression and antigenicity of DNA vaccines using a plasmid DNA construct expressing luciferase. Repeated intramuscular administration led to diminished luciferase expression, suggesting a role for immune‐mediated clearance of expression. To investigate the role of cell trafficking, we used the sphingosine 1‐phosphate receptor (S1PR) modulator, FTY720 (Fingolimod), which traps lymphocytes within the lymphoid tissues. When lymphocyte trafficking was blocked with FTY720, DNA transgene expression was maintained at a constant level for a significantly extended time period. Both continuous and staggered administration of FTY720 prolonged transgene expression. However, blocking lymphocyte egress during primary transgene administration did not result in an increase of transgene expression during secondary administration. Interestingly, there was a disconnect between transgene expression and immunogenicity, as increasing expression by this approach did not enhance the overall immune response. Furthermore, when FTY720 was administered alongside a DNA vaccine expressing the HIV gp140 envelope antigen, there was a significant reduction in both antigen‐specific antibody and T‐cell responses. This indicates that the developing antigen‐specific cellular response clears DNA vaccine expression but requires access to the site of expression in order to develop an effective immune response.

Synthesis of fluorescence labelled aptamers for use as low-cost reagents in HIV/AIDS research and diagnostics.

Aptamers are nucleic acids selected by systematic evolution of ligands by exponential enrichment. They have potential as alternatives to antibodies in medical research and diagnostics, with the advantages of being non-immunogenic and relatively inexpensive to produce. In the present study, gp120 aptamers conjugated with fluorescein isothiocyanate (FITC) were generated, which could interact with HIV-1 gp120. A previously isolated gp120 aptamer, CSIR 1.1, was conjugated with FITC by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and imidazole. The conjugation and binding to the glycoprotein were confirmed by flow cytometry. FITC conjugated aptamers showed an increase in fluorescence emission 24-fold higher than baseline, and this difference was statistically significant (P=0.0016). Compared with a commercially available biotinylated anti-gp120 antibody, detected using FITC conjugated streptavidin, the emission of fluorescence obtained from the FITC-conjugated aptamer was 8-fold higher, suggesting a stronger interaction with gp120. In addition, the FITC conjugated aptamer neutralized HIV-1 pseudoviruses with an average IC50 of 21.3 nM, similar to the parent aptamer that had an IC50 of 19.2 nM. However, the difference in inhibition between the two aptamers was not statistically significant (P=0.784). These results indicate that the FITC-conjugated aptamer generated in the present study could potentially be used as a low-cost reagent in HIV/AIDS research and diagnostics.

A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration

Despite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking β1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

Chemoenzymatic Synthesis and Antibody Binding of HIV-1 V1/V2 Glycopeptide-Bacteriophage Qβ Conjugates as a Vaccine Candidate.

The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qβ conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qβ), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qβ-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qβ conjugates synthesized represent a promising candidate of HIV-1 vaccine.

Design, Synthesis, and Antiviral activity of 1,2,3,4-Tetrahydropyrimidine derivatives acting as novel entry inhibitors to target at “Phe43 cavity” of HIV-1 gp120

The HIV-1 invasion is initiated with the interaction of viral glycoprotein gp120 and cellular receptor CD4. The binding mechanism reveals two major hotspots involved in gp120-CD4 interaction. The first one is a hydrophobic cavity (Phe43 cavity) on gp120 capped with phenyl ring of phe43CD4 and the second is the electrostatic interaction between positive charge of Arg59CD4 and negative charge of Asp368gp120. Targeting these hotspots, small molecules for entry inhibition and HIV-1 neutralization were designed and tested. In the process, pyrimidine derivatives were identified as potent molecules to intercept gp120-CD4 binding by targeting both the hotspots. Herein, the synthesis, characterization of 1,2,3,4-Tetrahydropyrimidine derivatives, and biological evaluation on 93IN101, a clade C virus are presented. The paper presents a novel set of entry inhibitors to target dual hotspots on gp120 to inhibit protein-protein interactions.

HIV-1 gp120 Antagonists Also Inhibit HIV-1 Reverse Transcriptase by Bridging the NNRTI and NRTI Sites.

HIV-1 infection is typically treated using ≥2 drugs, including at least one HIV-1 reverse transcriptase (RT) inhibitor. Drugs targeting RT comprise nucleos(t)ide RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NRTI-triphosphates bind at the polymerase active site and, following incorporation, inhibit DNA elongation. NNRTIs bind at an allosteric pocket ∼10 Å away from the polymerase active site. This study focuses on compounds ("NBD derivatives") originally developed to bind to HIV-1 gp120, some of which inhibit RT. We have determined crystal structures of three NBD compounds in complex with HIV-1 RT, correlating with RT enzyme inhibition and antiviral activity, to develop structure-activity relationships. Intriguingly, these compounds bridge the dNTP and NNRTI-binding sites and inhibit the polymerase activity of RT in the enzymatic assays (IC50 < 5 μM). Two of the lead compounds, NBD-14189 and NBD-14270, show potent antiviral activity (EC50 < 200 nM), and NBD-14270 shows low cytotoxicity (CC50 > 100 μM).

Design of gp120 HIV-1 entry inhibitors by scaffold hopping via isosteric replacements.

We present the development of alternative scaffolds and validation of their synthetic pathways as a tool for the exploration of new HIV gp120 inhibitors based on the recently discovered inhibitor of this class, NBD-14136. The new synthetic routes were based on isosteric replacements of the amine and acid precursors required for the synthesis of NBD-14136, guided by molecular modeling and chemical feasibility analysis. To ensure that these synthetic tools and new scaffolds had the potential for further exploration, we eventually tested few representative compounds from each newly designed scaffold against the gp120 inhibition assay and cell viability assays.

Interactions of HIV gp41's membrane-proximal external region and transmembrane domain with phospholipid membranes from 31P NMR.

HIV-1 entry into cells requires coordinated changes of the conformation and dynamics of both the fusion protein, gp41, and the lipids in the cell membrane and virus envelope. Commonly proposed features of membrane deformation during fusion include high membrane curvature, lipid disorder, and membrane surface dehydration. The virus envelope and target cell membrane contain a diverse set of phospholipids and cholesterol. To dissect how different lipids interact with gp41 to contribute to membrane fusion, here we use 31P solid-state NMR spectroscopy to investigate the curvature, dynamics, and hydration of POPE, POPC and POPS membranes, with and without cholesterol, in the presence of a peptide comprising the membrane proximal external region (MPER) and transmembrane domain (TMD) of gp41. Static 31P NMR spectra indicate that the MPER-TMD induces strong negative Gaussian curvature (NGC) to the POPE membrane but little curvature to POPC and POPC:POPS membranes. The NGC manifests as an isotropic peak in the static NMR spectra, whose intensity increases with the peptide concentration. Cholesterol inhibits the NGC formation and stabilizes the lamellar phase. Relative intensities of magic-angle spinning 31P cross-polarization and direct-polarization spectra indicate that all three phospholipids become more mobile upon peptide binding. Finally, 2D 1H-31P correlation spectra show that the MPER-TMD enhances water 1H polarization transfer to the lipids, indicating that the membrane surfaces become more hydrated. These results suggest that POPE is an essential component of the high-curvature fusion site, and lipid dynamic disorder is a general feature of membrane restructuring during fusion.

Ezrin Peptide Therapy from HIV to COVID: Inhibition of Inflammation and Amplification of Adaptive Anti-Viral Immunity.

Human Ezrin Peptides (HEPs) are inhibitors of expression of IL-6 and other inflammatory cytokines, amplifiers of adaptive B cell and T cell immunity and enhancers of tissue repair. The mutation stable C-terminus of HIV gp120, mimics 69% of the “Hep-receptor”, a zipped α-helical structure in the middle of the α domain of human ezrin protein. Synthetic peptides homologous to the Hep-receptor of ezrin of five to fourteen amino acids, activate anti-viral immunity against a wide range of viruses (HIV, HCV, herpes, HPV, influenza and other human respiratory viruses). Human Ezrin Peptide One (HEP1) TEKKRRETVEREKE (brand name Gepon, registered for human use in Russia from 2001) is a successful treatment for opportunistic infections in HIV-infected patients. That treats HEP1and prevents mucosal candidiasis, herpes zoster outbreaks and infection-induced chronic diarrhea. There are clinical publications in Russian on the successful treatments of chronic recurrent vaginal candidiasis, acute and chronic enterocolitis and dysbacteriosis, which are accompanied by normalization of the mucosal microbiome, and the decline or disappearance of inflammation. HEP1 is also an effective treatment and prevention for recurrent inflammation and ulceration in the stomach, duodenum and colon. HEP1 and RepG3 GEKKRRETVEREGG (a derivative of HEP1) have been used successfully as an inhaled spray peptide solution to treat a small number of human volunteers with mild-to-moderate COVID, resulting from SARS-CoV-2 infection, based on earlier successes in treating acute viral respiratory disease with inflammatory complications. Ezrin peptides seem to correct a dysregulation of innate immune responses to SARS-CoV-2. They are also adjuvants of B cell adaptive immunity and increase antibody titres, resulting in protection from lethal virus infection of mice. In a clinical study in Moscow, orally administered HEP1 was shown to enhance antibody-titres produced in response to hepatitis-B vaccination. These very preliminary but promising results with ezrin peptide treatment of COVID must be replicated in large-scale randomised placebo controlled clinical studies, to be verified.

Conformational flexibility of the conserved hydrophobic pocket of HIV-1 gp41. Implications for the discovery of small-molecule fusion inhibitors

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene-1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP.

Design and Identification of Potential HIV-1 Entry Inhibitors Using In Silico Click Chemistry and Molecular Modeling Methods

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to computer-aided design of potential HIV-1 inhibitors able to block the membrane-proximal external region (MPER) of HIV-1 gp41, which plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds exhibiting high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Modeling HIV-1 Within-Host Dynamics After Passive Infusion of the Broadly Neutralizing Antibody VRC01.

VRC01 is a broadly neutralizing antibody that targets the CD4 binding site of HIV-1 gp120. Passive administration of VRC01 in humans has assessed the safety and the effect on plasma viremia of this monoclonal antibody (mAb) in a phase 1 clinical trial. After VRC01 infusion, the plasma viral load in most of the participants was reduced but had particular dynamics not observed during antiretroviral therapy. In this paper, we introduce different mathematical models to explain the observed dynamics and fit them to the plasma viral load data. Based on the fitting results we argue that a model containing reversible Ab binding to virions and clearance of virus-VRC01 complexes by a two-step process that includes (1) saturable capture followed by (2) internalization/degradation by phagocytes, best explains the data. This model predicts that VRC01 may enhance the clearance of Ab-virus complexes, explaining the initial viral decay observed immediately after antibody infusion in some participants. Because Ab-virus complexes are assumed to be unable to infect cells, i.e., contain neutralized virus, the model predicts a longer-term viral decay consistent with that observed in the VRC01 treated participants. By assuming a homogeneous viral population sensitive to VRC01, the model provides good fits to all of the participant data. However, the fits are improved by assuming that there were two populations of virus, one more susceptible to antibody-mediated neutralization than the other.

Variability in the Glycosylation Patterns of gp120 Proteins from Different Human Immunodeficiency Virus Type 1 Isolates Expressed in Different Host Cells.

The mature HIV-1 envelope (Env) glycoprotein is composed of gp120, the exterior subunit, and gp41, the transmembrane subunit assembled as trimer by noncovalent interaction. There is a great body of literature to prove that gp120 binds to CD4 first, then to the coreceptor. Binding experiments and functional assays have demonstrated that CD4 binding induces conformational changes in gp120 that enable or enhance its interaction with a coreceptor. Previous studies provided different glycomic maps for the HIV-1 gp120. Here, we build on previous work to report that the use of LC-MS/MS, in conjunction with hydrophilic interaction liquid chromatography (HILIC) enrichment to glycosylation sites, associated with the assorted neutralizing or binding events of glycosylation targeted antibodies from different clades or strains. In this study, the microheterogeneity of the glycosylation from 4 different clades of gp120s is deeply investigated. Aberrant glycosylation patterns were detected on gp120 that originated from different clades, viral sequences, and host cells. The results of this study may help provide a better understanding of the mechanism of how the glycans participate in the antibody neutralizing process that targets glycosylation sites.

Modulation of OPRM1 Alternative Splicing by Morphine and HIV-1 Nef.

Clinically used opioids, such as morphine, activate the mu opioid receptor (MOR) encoded by Opioid Receptor Mu 1 (OPRM1) gene. Examination of the opioid receptor genes showed that the human OPRM1 pre-mRNA undergoes extensive alternative splicing events and capable of expressing 21 isoforms. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR-1) has been extensively studied. Compounding this issue is the increasing significance of intravenous drug abuse in HIV neuropathogenesis. Here, we investigated the molecular impact of morphine and HIV-1 on regulation of OPRM1 pre-mRNA splicing in in vitro and in vivo models. Our results suggested that morphine treatment specifically induces the alternative splicing of MOR-1X isoform among the other isoforms analyzed in neuronal cells. Interestingly, alternative splicing and expression of MOR-1X isoform was also induced in postmortem brain tissues obtained from people with HIV (PWH). Additionally, treatment of control rats with morphine induced alternative splicing of MOR-1X in the brain regions involved in the reward pathways. More interestingly, HIV-1 transgenic (HIV-1Tg) rats, showed an additive induction of MOR-1X isoform with the exposure to morphine. To further assess the possible role of HIV secretory proteins in alternative splicing of OPRM1 gene, we analyzed the impact of HIV-1 Tat, gp120 and Nef proteins on alternative splicing of MOR-1X isoform. While the Tat and gp120 had no visible effects, treatment of neurons with Nef induced MOR-1X alternative splicing that was comparable to treatment with morphine. Altogether, our results suggest that HIV-1 may alter MOR isoform expression with Nef protein by amplifying the rate of MOR-1X alternative splicing induced by morphine.

The Chemokine CCL5 Inhibits the Replication of Influenza A Virus Through SAMHD1 Modulation.

Influenza A virus (IAV) is the main etiological agent of acute respiratory tract infections. During IAV infection, interferon triggers the overexpression of restriction factors (RFs), the intracellular antiviral branch of the innate immune system. Conversely, severe influenza is associated with an unbalanced pro-inflammatory cytokine release. It is unclear whether other cytokines and chemokines released during IAV infection modulate RFs to control virus replication. Among the molecules enhanced in the infected respiratory tract, ligands of the CCR5 receptor play a key role, as they stimulate the migration of inflammatory cells to the alveoli. We investigated here whether ligands of the CCR5 receptor could enhance RFs to levels able to inhibit IAV replication. For this purpose, the human alveolar basal epithelial cell line (A549) was treated with endogenous (CCL3, CCL4 and CCL5) or exogenous (HIV-1 gp120) ligands prior to IAV infection. The three CC-chemokines tested reduced infectious titers between 30% to 45% upon 24 hours of infection. Eploying RT-PCR, a panel of RF mRNA levels from cells treated with CCR5 agonists was evaluated, which showed that the SAMHD1 expression was up-regulated four times over control upon exposure to CCL3, CCL4 and CCL5. We also found that IAV inhibition by CCL5 was dependent on PKC and that SAMHD1 protein levels were also increased after treatment with CCL5. In functional assays, we observed that the knockdown of SAMHD1 resulted in enhanced IAV replication in A549 cells and abolished both CCL5-mediated inhibition of IAV replication and CCL5-mediated cell death inhibition. Our data show that stimuli unrelated to interferon may trigger the upregulation of SAMHD1 and that this RF may directly interfere with IAV replication in alveolar epithelial cells.

A large repertoire of B cell lineages targeting one cluster of epitopes in a vaccinated rhesus macaque

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10−5 at week 10 that declined to 10−3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10−5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the Cterminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.