Identification of a mimotope of a complex gp41 Human Immunodeficiency VIrus epitope related to a non-structural protein of Hepacivirus previously implicated in Kawasaki disease.

Background: We have previously isolated a highly mutated VH1-02 antibody termed group C 76-Q13-6F5 (6F5) that targets a conformational epitope on gp41. 6F5 has the capacity to mediate Ab dependent cell cytotoxicity (ADCC). When the VH1-02 group C 76 antibodies variable chain sequence was reverted to germline (76Canc), this still retained ADCC activity. Due to this ability for the 76Canc germline antibody to functionally target this epitope, we sought to identify a protein target for vaccine development. Methods: Initially, we interrogated peptide targeting by screening a microarray containing 29,127 linear peptides. Western blot and ELISAs were used to confirm binding and explore human serum targeting. Autoimmune targeting was further interrogated on a yeast-displayed human protein microarray. Results: 76Canc specifically recognized a number of acidic peptides. Meme analysis identified a peptide sequence similar to a non-structural protein of Hepacivirus previously implicated in Kawasaki disease (KD). Binding was confirmed to top peptides, including the Hepacivirus-related and KD-related peptide. On serum competitions studies using samples from children with KD compared to controls, targeting of this epitope showed no specific correlation to having KD. Human protein autoantigen screening was also reassuring. Conclusions: This study identifies a peptide that can mimic the gp41 epitope targeted by 76C group antibodies (i.e. a mimotope). We show little risk of autoimmune targeting including any inflammation similar to KD, implying non-specific targeting of this peptide during KD. Development of such peptides as the basis for vaccination should proceed cautiously.

Structure and Interactions of HIV-1 gp41 CHR-NHR Reverse Hairpin Constructs Reveal Molecular Determinants of Antiviral Activity.

Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.

Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems.

The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.

Int&in: A machine learning-based web server for active split site identification in inteins.

Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. In nature, inteins are either contiguous or split. In the case of split inteins, the two fragments must first form a complex for the splicing to occur. Contiguous inteins have previously been artificially split in two fragments because split inteins allow for distinct applications than contiguous ones. Even naturally split inteins have been split at unnatural split sites to obtain fragments with reduced affinity for one another, which are useful to create conditional inteins or to study protein-protein interactions. So far, split sites in inteins have been heuristically identified. We developed Int&in, a web server freely available for academic research (https://intein.biologie.uni-freiburg.de) that runs a machine learning model using logistic regression to predict active and inactive split sites in inteins with high accuracy. The model was trained on a dataset of 126 split sites generated using the gp41-1, Npu DnaE and CL inteins and validated using 97 split sites extracted from the literature. Despite the limited data size, the model, which uses various protein structural features, as well as sequence conservation information, achieves an accuracy of 0.79 and 0.78 for the training and testing sets, respectively. We envision Int&in will facilitate the engineering of novel split inteins for applications in synthetic and cell biology.

HIV-1 envelope facilitates the development of protease inhibitor resistance through acquiring mutations associated with viral entry and immune escape.

Introduction: There is increasing evidence supporting a role for HIV-1 envelope in the development of Protease Inhibitor drug resistance, and a recent report from our group suggested that Env mutations co-evolve with Gag-Protease mutations in the pathway to Lopinavir resistance. In this study, we investigated the effect of co-evolving Env mutations on virus function and structure. Methods: Co-receptor usage and n-linked glycosylation were investigated using Geno2Pheno as well as tools available at the Los Alamos sequence database. Molecular dynamics simulations were performed using Amber 18 and analyzed using Cpptraj, and molecular interactions were calculated using the Ring server. Results: The results showed that under Protease Inhibitor drug selection pressure, the envelope gene modulates viral entry by protecting the virus from antibody recognition through the increased length and number of N-glycosylation sites observed in V1/V2 and to some extent V5. Furthermore, gp120 mutations appear to modulate viral entry through a switch to the CXCR4 coreceptor, induced by higher charge in the V3 region and specific mutations at the coreceptor binding sites. In gp41, S534A formed a hydrogen bond with L602 found in the disulfide loop region between the Heptad Repeat 1 and Heptad Repeat 2 domains and could negatively affect the association of gp120-gp41 during viral entry. Lastly, P724Q/S formed both intermolecular and intramolecular interactions with residues within the Kennedy loop, a known epitope. Discussion: In conclusion, the results suggest that mutations in envelope during Protease Inhibitor treatment failure are related to immune escape and that S534A mutants could preferentially use the cell-to-cell route of infection.

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Frequency of Fusion Inhibitor Resistance Mutations Among Therapy-Naïve HIV Patients.

Glycoprotein 41 (gp41) of the human immunodeficiency virus type 1 (HIV-1) protein plays a critical role in membrane fusion. Gp41 binds to proteins in the plasma membrane of CD4+ T cells, particularly the T-cell antigen receptor (TCR). These findings indicate that gp41 is involved in the assembly of HIV-1 at the plasma membrane of T cells and affects the stimulation of the TCR. To control HIV-1, new inhibitors were introduced to target the gp41 protein. However, mutations in this region might reduce their efficacy. The Gp41 region was amplified from the sera of 30 patients using nested polymerase chain reaction. The sequences were analyzed by bioinformatics tools to identify mutations and gp41 structural features. Subtyping and the interaction between fusion inhibitors and gp41 proteins were also examined. As the first report from Iran, docking analysis between fusion inhibitors and Iranian gp41 proteins showed that mutations in gp41 could not reduce the efficacy of the fusion inhibitors. Most of the patients were infected with CRF35-AD. Several post-modification positions, including glycosylation and phosphorylation sites, were identified in the gp41 protein. Our findings revealed no known multinational drug resistance to gp41 inhibitors; thus, fusion inhibitors can effectively inhibit HIV in Iranian patients. In addition, the present study introduced a new gp41 region (36-44 aa), which considerably influences the interactions between gp41 inhibitors and the gp41 protein. This region may play a pivotal role in suppressing gp41 inhibitors in CFR35-AD. Furthermore, gp41 can be considered a good target for subtyping analysis via the phylogenetic method.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Molecular dynamics simulations show how antibodies may rescue HIV-1 mutants incapable of infecting host cells.

High mutation and replication rates of HIV-1 result in the continuous generation of variants, allowing it to adapt to changing host environments. Mutations often have deleterious effects, but variants carrying them are rapidly purged. Surprisingly, a particular variant incapable of entering host cells was found to be rescued by host antibodies targeting HIV-1. Understanding the molecular mechanism of this rescue is important to develop and improve antibody-based therapies. To unravel the underlying mechanisms, we performed fully atomistic molecular dynamics simulations of the HIV-1 gp41 trimer responsible for viral entry into host cells, its entry-deficient variant, and its complex with the rescuing antibody. We find that the Q563R mutation, which the entry-deficient variant carries, prevents the native conformation of the gp41 6-helix bundle required for entry and stabilizes an alternative conformation instead. This is the consequence of substantial changes in the secondary structure and interactions between the domains of gp41. Binding of the antibody F240 to gp41 reverses these changes and re-establishes the native conformation, resulting in rescue. To test the generality of this mechanism, we performed simulations with the entry-deficient L565A variant and antibody 3D6. We find that 3D6 binding was able to reverse structural and interaction changes introduced by the mutation and restore the native gp41 conformation. Viral variants may not only escape antibodies but be aided by them in their survival, potentially compromising antibody-based therapies, including vaccination and passive immunization. Our simulation framework could serve as a tool to assess the likelihood of such resistance against specific antibodies.Communicated by Ramaswamy H. SarmaCommunicated by Ramaswamy H. Sarma.

Development of Zika Virus E Variants for Pseudotyping Retroviral Vectors Targeting Glioblastoma Cells.

A fundamental idea for targeting glioblastoma cells is to exploit the neurotropic properties of Zika virus (ZIKV) through its two outer envelope proteins, prM and E. This study aimed to develop envelope glycoproteins for pseudotyping retroviral vectors that can be used for efficient tumor cell infection. Firstly, the retroviral vector pNLlucAM was packaged using wild-type ZIKV E to generate an E-HIVluc pseudotype. E-HIVluc infection rates for tumor cells were higher than those of normal prME pseudotyped particles and the traditionally used vesicular stomatitis virus G (VSV-G) pseudotypes, indicating that protein E alone was sufficient for the formation of infectious pseudotyped particles. Secondly, two envelope chimeras, E41.1 and E41.2, with the E wild-type transmembrane domain replaced by the gp41 transmembrane and cytoplasmic domains, were constructed; pNLlucAM or pNLgfpAM packaged with E41.1 or E41.2 constructs showed infectivity for tumor cells, with the highest rates observed for E41.2. This envelope construct can be used not only as a tool to further develop oncolytic pseudotyped viruses for therapy, but also as a new research tool to study changes in tumor cells after the transfer of genes that might have therapeutic potential.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

An Artificial Peptide-Based Bifunctional HIV-1 Entry Inhibitor That Interferes with Viral Glycoprotein-41 Six-Helix Bundle Formation and Antagonizes CCR5 on the Host Cell Membrane.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high variability and drug resistance. This has necessitated the development of antivirals with a new chemotype and therapy. We previously identified an artificial peptide with non-native protein sequence, AP3, with the potential to inhibit HIV-1 fusion through targeting hydrophobic grooves on the N-terminal heptad repeat trimer of viral glycoprotein gp41. Here, a small-molecule HIV-1 inhibitor targeting chemokine coreceptor CCR5 on the host cell was integrated into the AP3 peptide, producing a novel dual-target inhibitor with improved activity against multiple HIV-1 strains including those resistant to the currently used anti-HIV-1 drug enfuvirtide. Its superior antiviral potency in comparison with the respective pharmacophoric moieties is in consonance with the dual binding of viral gp41 and host factor CCR5. Therefore, our work provides a potent artificial peptide-based bifunctional HIV-1 entry inhibitor and highlights the multitarget-directed ligands approach in the development of novel therapeutic anti-HIV-1 agents.

Identification of Anti-gp41 Monoclonal Antibodies That Effectively Target Cytotoxic Immunoconjugates to Cells Infected with Human Immunodeficiency Virus, Type 1.

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs.

Multitargeted drug design strategy for discovery of short-peptide-based HIV-1 entry inhibitors with high potency.

The development of short-peptide-based inhibitors to prevent HIV-1 entry into the host cell has been rewarded with limited success. Herein, we report a multitarget-directed ligand strategy to generate a series of short-peptide HIV-1 entry inhibitors that integrated the pharmacological activities of a peptide fusion inhibitor able to disrupt HIV-1 gp41 glycoprotein hexameric coiled-coil assembly and a small-molecule CCR5 antagonist that blocks the interaction between HIV-1 and its coreceptor. Among these inhibitors, dual-target 23-residue peptides SP12T and SP12L displayed dramatically increased inhibitory activities against HIV-1 replication as compared to the marketed 36-residue peptide T20. Moreover, results suggested that SP12T and SP12L successfully performed a dual-targeting mechanism. It can be concluded that these short-peptide-based HIV-1 entry inhibitors have potential for further development as candidates for a novel multitarget therapy to treat HIV-1 infection.

Structure-guided stabilization improves the ability of the HIV-1 gp41 hydrophobic pocket to elicit neutralizing antibodies.

The hydrophobic pocket found in the N-heptad repeat (NHR) region of HIV-1 gp41 is a highly conserved epitope that is the target of various HIV-1-neutralizing monoclonal antibodies. Although the high conservation of the pocket makes it an attractive vaccine candidate, it has been challenging to elicit potent anti-NHR antibodies via immunization. Here, we solved a high-resolution structure of the NHR mimetic IQN17, and, consistent with previous ligand-bound gp41 pocket structures, we observed remarkable conformational plasticity of the pocket. The high malleability of this pocket led us to test whether we could improve the immunogenicity of the gp41 pocket by stabilizing its conformation. We show that the addition of five amino acids at the C terminus of IQN17, to generate IQN22, introduces a stabilizing salt bridge at the base of the peptide that rigidifies the pocket. Mice immunized with IQN22 elicited higher avidity antibodies against the gp41 pocket and a more potent, albeit still weak, neutralizing response against HIV-1 compared with IQN17. Stabilized epitope-focused immunogens could serve as the basis for future HIV-1 fusion-inhibiting vaccines.

A large HIV gp41 construct with trimer-of-hairpins structure exhibits V2E mutation-dominant attenuation of vesicle fusion and helicity very similar to V2E attenuation of HIV fusion and infection and supports: (1) hairpin stabilization of membrane apposition with larger distance for V2E; and (2) V2E dominance by an antiparallel ß sheet with interleaved fusion peptide strands from two gp41 trimers.

There is complete attenuation of fusion and infection mediated by HIV gp160 with gp41 subunit with V2E mutation, and also V2E dominance with WT/V2E mixtures. V2E is at the N-terminus of the ∼25-residue fusion peptide (Fp) which likely binds the target membrane. In this study, large V2E attenuation and dominance were observed for vesicle fusion induced by FP_HM, a large gp41 ectodomain construct with Fp followed by hyperthermostable hairpin with N- and C-helices, and membrane-proximal external region (Mper). FP_HM is a trimer-of-hairpins, the final gp41 structure during fusion. Vesicle fusion and helicity were measured for FP_HM using trimers with different fractions (f's) of WT and V2E proteins. Reductions in FP_HM fusion and helicity vs. fV2E were quantitatively-similar to those for gp160-mediated fusion and infection. Global fitting of all V2E data supports 6 WT gp41 (2 trimers) required for fusion. These data are understood by a model in which the ∼25 kcal/mol free energy for initial membrane apposition is compensated by the thermostable hairpin between the Fp in target membrane and Mper/transmembrane domain in virus membrane. The data support a structural model for V2E dominance with a membrane-bound Fp with antiparallel ß sheet and interleaved strands from the two trimers. Relative to fV2E = 0, a longer Fp sheet is stabilized with small fV2E because of salt-bridge and/or hydrogen bonds between E2 on one strand and C-terminal Fp residues on adjacent strands, like R22. A longer Fp sheet results in shorter N- and C-helices, and larger separation during membrane apposition which hinders fusion.

Enhancing HIV-1 Neutralization by Increasing the Local Concentration of Membrane-Proximal External Region-Directed Broadly Neutralizing Antibodies.

Broadly neutralizing antibodies (bNAbs) against the membrane-proximal external region (MPER) of the gp41 component of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) are characterized by long, hydrophobic, heavy chain complementarity-determining region 3s (HCDR3s) that interact with the MPER and some viral membrane lipids to achieve increased local concentrations. Here, we show that increasing the local concentration of MPER-directed bNAbs at the cell surface via binding to the high-affinity Fc receptor FcγRI potentiates their ability to prevent viral entry in a manner analogous to the previously reported observation wherein the lipid-binding activity of MPER bNAbs increases their concentration at the viral surface membrane. However, binding of MPER-directed bNAb 10E8 to FcγRI abolishes the neutralization synergy that is seen with the N-heptad repeat (NHR)-targeting antibody D5_AR and NHR-targeting small molecule enfuvirtide (T20), possibly due to decreased accessibility of the NHR in the FcγRI-10E8-MPER complex. Taken together, our results suggest that lipid-binding activity and FcγRI-mediated potentiation function in concert to improve the potency of MPER-directed bNAbs by increasing their local concentration near the site of viral fusion. Therefore, lipid binding may not be a strict requirement for potent neutralization by MPER-targeting bNAbs, as alternative methods can achieve similar increases in local concentrations while avoiding potential liabilities associated with immunologic host tolerance. IMPORTANCE The trimeric glycoprotein Env, the only viral protein expressed on the surface of HIV-1, is the target of broadly neutralizing antibodies and the focus of most vaccine development efforts. Broadly neutralizing antibodies targeting the membrane proximal external region (MPER) of Env show lipid-binding characteristics, and modulating this interaction affects neutralization. In this study, we tested the neutralization potencies of variants of the MPER-targeting antibody 10E8 with different viral-membrane-binding and host FcγRI-binding capabilities. Our results suggest that binding to both lipid and FcγRI improves the neutralization potency of MPER-directed antibodies by concentrating the antibodies at sites of viral fusion. As such, lipid binding may not be uniquely required for MPER-targeting broadly neutralizing antibodies, as alternative methods to increase local concentration can achieve similar improvements in potency.

Chromatographic single-step purification of tagless proteins using gp41-1 split inteins.

The current trend in biopharmaceutical drug manufacturing is towards increasing potency and complexity of products such as peptide scaffolds, oligonucleotides and many more. Therefore, a universal affinity purification step is important in order to meet the requirements for cost and time efficient drug production. By using a self-splicing intein affinity tag, a purification template is generated that allows for a universal chromatographic affinity capture step to generate a tagless target protein without the use of proteases for further tag removal. This study describes the successful implementation of gp41-1-based split inteins in a chromatographic purification process for, e.g., E. coli-derived targets. The tagless target is generated in a single-step purification run. The on-column cleavage is induced by triggering a simple pH change in the buffer conditions without the need for additives such as Zn2+ or thiols. This system has proven to be reusable for at least ten purification cycles that use 150 mM H3PO4 as the cleaning agent.

Targeting a Conserved Lysine in the Hydrophobic Pocket of HIV-1 gp41 Improves Small Molecule Antiviral Activity.

Human Immunodeficiency virus (HIV-1) fusion is mediated by glycoprotein-41, a protein that has not been widely exploited as a drug target. Small molecules directed at the gp41 ectodomain have proved to be poorly drug-like, having moderate efficacy, high hydrophobicity and/or high molecular weight. We recently investigated conversion of a fairly potent hydrophobic inhibitor into a covalent binder, by modifying it to react with a lysine residue on the protein. We demonstrated a 10-fold improvement in antiviral efficacy. Here, we continue this study, utilizing instead molecules with better inherent drug-like properties. Molecules possessing low to no antiviral activity as equilibrium binders were converted into µM inhibitors upon addition of an electrophilic warhead in the form of a sulfotetrafluorophenyl (STP) activated ester. We confirmed specificity for gp41 and for entry. The small size of the inhibitors described here offers an opportunity to expand their reach into neighboring pockets while retaining drug-likeness. STP esterification of equilibrium binders is a promising avenue to explore for inhibiting HIV-1 entry. Many gp41 targeting molecules studied over the years possess carboxylic acid groups which can be easily converted into the corresponding STP ester. It may be worth the effort to evaluate a library of such inhibitors as a way forward to small molecule inhibition of fusion of HIV and possibly other enveloped viruses.

Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.