A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

Emerging Role of Calcium-Activated Potassium Channel in the Regulation of Cell Viability Following Potassium Ions Challenge in HEK293 Cells and Pharmacological Modulation

Emerging evidences suggest that Ca²⁺activated-K⁺-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10^-11-10^-3M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2x10^-7M-5x10^-3M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K⁺ ion homeostasis including periodic paralysis and myotonia.     

A Disintegrin and Metalloprotease (ADAM) 10 and ADAM17 Are Major Sheddases of T Cell Immunoglobulin and Mucin Domain 3 (Tim-3)

T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4⁺ and CD8⁺ effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu¹⁸¹–Asp¹⁹⁰ of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14⁺ monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14⁺ monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14⁺ monocytes.     

Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.It was recently reported that TZM-bl cells, which are commonly used to assess neutralizing antibodies (Abs) against human immunodeficiency virus type 1 (HIV-1), are contaminated with an ecotropic murine leukemia virus (MLV) (22). TZM-bl (also called JC.53bl-13) is a HeLa cell derivative that was engineered by amphotropic retroviral transduction to express CD4 and CCR5 (17) and was further engineered with an HIV-1-based vector to contain Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli β-galactosidase (24). These engineered features made TZM-bl cells highly susceptible to HIV-1 infection in a readily quantifiable assay for neutralizing Abs. Many published studies used this cell line for assessments of HIV-1 neutralization; these include several recent reports describing the magnitude, breadth, and epitope specificity of the neutralizing Ab response in infected individuals (14, 18-20), neutralization escape (25), and the neutralization phenotype of transmitted/founder viruses (10). TZM-bl cells are also gaining popularity for assessments of vaccine-elicited neutralizing Ab responses (13). The validity of these and other published results, together with a rationale for the continued use of TZM-bl cells in assessing neutralizing Abs against HIV-1, are very dependent on establishing to what extent, if any, MLV contamination affects the outcome of the assay.It was suggested that ecotropic MLV entered TZM-bl cells via the progenitor JC.53 cell line as an amphotropic MLV pseudotype (22). In this regard, JC.53 cells were constructed from HeLa cells in two stages by using ping-pong technology to amplify the pSFF vector derived from the replication-defective and highly truncated Friend spleen focus-forming virus (3). When used with this vector, this procedure has previously resulted in stable vector expression (17) without formation of replication-competent MLV recombinants (8, 11). A panel of HeLa-CD4 clones was made that express different amounts of CD4 and where the high-expression HI-J clone was used to make a derivative panel of clones (termed JC), including JC.53, that expressed diverse levels of CCR5 (9, 16, 17). In addition, the HeLa-CD4 clone HI-R that expressed low levels of CD4 was used to make another panel of CCR5-expressing clones (termed RC). To investigate this newly reported issue, cell extracts from these clonal panels and from TZM-bl cells were analyzed for MLV Gag antigens by Western immunoblotting. Representative data, as shown in Fig. ​Fig.1A,1A, confirm that JC.53 and TZM-bl cells express MLV Gag antigens, whereas the progenitor HI-J clone of HeLa-CD4 cells and many but not all of the other HeLa-CD4/CCR5 clones in the JC panel lack MLV antigens.Open in a separate windowFIG. 1.Characterization of HeLa clones for MLV Gag expression, HIV-1 susceptibility, and cell surface expression of HIV-1 fusion receptors. (A) MLV Gag antigen expression in HeLa cells and derivative clones expressing CD4 or CD4 and CCR5. Cell lysates were prepared from the cell clones and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with Abs to MLV Gag antigens (upper blot). The lysates were also probed with anti-tubulin antibodies (lower blot). Lane 1, HeLa cells; lanes 2 and 3, HeLa CD4 clones HI-R and HI-J, respectively; lanes 4, 5, and 6, HeLa-CD4/CCR5 clones JC.10, JC.48, and JC.53, respectively; lane 7, TZM-bl cells; lane 8, psi-2 packaging cells positive for MLV Gag. (B) HIV-1 infectivity on the HeLa-CD4/CCR5 JC panel. Target cells were infected with HIV-1 isolate JRCSF that had been produced from clone JC.53 cells (black) or with JRCSF produced from transfected HEK293T cells (red). The target cells were also infected with the JR-FL isolate produced from peripheral blood mononuclear cells (PBMC; green). The HeLa-CD4/CCR5 target cells had a CCR5 expression range of 2 × 10³ (clone JC.10) to 1.3 × 10⁵ (clones JC.53 and TZM-bl) CCR5 molecules/cell. Each set of three data points at a given CCR5 expression level represents a single HeLa-CD4/CCR5 JC clone. None of the HIV-1 isolates was able to infect HeLa-CD4 cells lacking CCR5. The blue asterisks indicate clones that are negative for MLV Gag proteins. Clones JC.48 (used for subsequent infection and neutralization assays) and JC.53 (progenitor of TZM-bl cells) are specifically labeled. (C) Surface expression of CD4, CCR5, and CXCR4 on TZM-bl and JC.48 cells was assessed by flow cytometry using the same stocks of cells that were used in infection and neutralization assays in Fig. ​Fig.2.2. Surface staining was performed with phycoerythrin-conjugated mouse monoclonal Abs to CD4, CCR5 (CD195), and CXCR4 (CD184). Background staining was performed with isotype-matched control Abs. All Abs for flow cytometry were purchased from BD Biosciences Pharmingen (San Diego, CA). Results are shown as the mean fluorescence intensity (MFI) of positive cells. Most cells (>90%) stained positive in each case.Initial studies of HI-R cells and other clonal panels that were made using these methods also suggested a lack of MLV antigens (data not shown). We then determined the titers of replication-competent HIV-1_JRCSF preparations using JC.53 and TZM-bl cells as well as other representative HeLa-CD4/CCR5 clones in the JC panel. The results are plotted in Fig. ​Fig.1B1B as a function of cellular CCR5 content. Clones having more than a low threshold level of ∼8,000 CCR5/cell were equally susceptible to infection regardless of whether they contained MLV antigens, clearly demonstrating that HIV-1_JRCSF titers were not significantly affected by MLV. As expected, titers obtained with JC.53 and TZM-bl cells were also equivalent. In addition, these results demonstrate that HIV-1_JRCSF preparations made in JC.53 cells and in cells lacking MLV antigens (i.e., HEK293T cells and human peripheral blood mononuclear cells) were unable to infect HeLa cells lacking CCR5. The results in Fig. ​Fig.1B1B were expected because previous studies demonstrated that ecotropic MLVs cannot infect human cells or even bind to the human CAT-1 receptor paralog (1, 6, 21, 23). Moreover, it has been shown that ecotropic host range MLVs do not interfere with superinfection by any retrovirus capable of infecting human cells, including gibbon ape leukemia virus, amphotropic MLV, baboon endogenous virus, and feline leukemia virus subgroup C (21). In view of the report by Takeuchi et al. (22), we were surprised to find that JC.53 and TZM-bl cells express very small amounts of ecotropic MLV Env glycoproteins, as indicated by immunofluorescence microscopy and by their resistance to complement-dependent killing by a cytotoxic antiserum specific for MLV envelope glycoproteins (6). Nevertheless, the cell clones that contained MLV Gag all released ecotropic host range virions that replicated in murine NIH 3T3 cells but not in human cells (data not shown).To determine whether MLV affects the measurement of neutralizing Abs in TZM-bl cells, parallel assays were performed in TZM-bl and JC.48 cells; these latter cells were determined to be MLV free by Western blot analysis (Fig. ​(Fig.1)1) and by an inability to transfer MLV infection to NIH 3T3 cells (data not shown). Because JC.48 cells express CCR5 at somewhat lower levels than JC.53 cells (∼2-fold lower; Fig. ​Fig.1B),1B), it may be expected that they would be less susceptible to HIV-1 infection than are TZM-bl cells. Differences in susceptibility to HIV-1 infection may require the use of adjusted virus doses to achieve equivalent assay performance when measuring neutralizing Abs. Indeed, levels of CD4 and CCR5 were approximately twofold lower on JC.48 cells than on TZM-bl cells, whereas levels of CXCR4 were approximately equal (Fig. ​(Fig.1C).1C). We therefore measured the susceptibility of both cell lines to infection by three molecularly cloned Env-pseudotyped viruses, each bearing an Env from a different CCR5-tropic HIV-1 subtype B virus (SF162.LS, Bal.26, and QH0692.42). Infection was quantified by Luc activity expressed as relative luminescence units (RLU). Because JC.48 cells do not contain a reporter gene, the Env-pseudotyped viruses were prepared by cotransfection with the NL4-3.Luc.R-E- reporter backbone plasmid (7). Identical Luc-containing, Env-pseudotyped virus stocks were used in both cell lines. As shown in Fig. ​Fig.2A,2A, the infectivity of each pseudotyped virus was somewhat diminished in JC.48 cells compared to the infectivity in TZM-bl cells. Nonetheless, the levels of infectivity in JC.48 cells remained acceptable for neutralization assays.Open in a separate windowFIG. 2.HIV-1 infectivity and neutralization in TZM-bl and JC.48.CD4.CCR5 cells. (A) TZM-bl and JC.48 cells were incubated with serial fourfold dilutions (11 dilutions total) of three HIV-1 Env-pseudotyped viruses in quadruplicate in 96-well culture plates. Luc activity was measured after 48 h of incubation and is expressed as RLU after subtraction of background luminescence from cell control wells. Squares, TZM-bl cells; triangles, JC.48 cells. (B) Neutralization assays were performed with three HIV-1 Env-pseudotyped viruses in either TZM-bl or JC.48 cells. Input virus doses were adjusted to yield equivalent infectivity in both cell lines. Black bars, TZM-bl; gray bars, JC.48. Top panel: sCD4, monoclonal Abs, and HIVIG (purified immunoglobulin G from pooled HIV-1-positive plasmas). Bottom panel: individual HIV-1-positive plasma samples. The same three stocks of virus were used in both experiments. All three Env-pseudotyped viruses were prepared with the NL4-3.Luc.R-E- reporter backbone plasmid.With this information in hand, neutralization assays were performed in JC.48 and TZM-bl cells using adjusted virus doses that yielded equivalent infectivity levels in both cell lines. These neutralization assays were performed in a 96-well format as described previously (12), where the 50% inhibitory dose (ID₅₀) was reported as either the concentration or sample dilution at which RLU were reduced by 50% compared to RLU in virus control wells (cells plus virus without test sample) after subtraction of background RLU from cell control wells (cells only). A wide variety of serologic reagents was tested, including sCD4, a monoclonal Ab to the CD4 binding site of gp120 (immunoglobulin G1b12) (15); a monoclonal Ab that recognizes a glycan-specific epitope on gp120 (2G12) (5); two monoclonal Abs that recognize adjacent epitopes in the membrane proximal external region of gp41 (2F5 and 4E10) (2, 4); and serum samples from seven antiretroviral-naive HIV-1-infected individuals. As shown in Fig. ​Fig.2B,2B, results in the two cell lines were similar for all three viruses and all serologic reagents tested. Indeed, ID₅₀ values in the two cell types agreed within twofold, which is within the normal range of variability of the assay. These results indicate that equivalent neutralization results were obtained in both cell lines.In summary, we found no evidence that ecotropic MLV contamination in TZM-bl cells has a measurable effect on HIV-1 neutralization when these cells are used as targets for infection. This outcome indicates that the presence of ecotropic MLV in TZM-bl cells does not alter the ability of Ab to neutralize HIV-1, nor does it interfere with the detection of neutralization by using HIV-1 Tat-regulated reporter gene expression in a single-cycle infection assay. However, we discourage the use of TZM-bl cells to generate HIV-1 stocks, because the latter would likely be contaminated with ecotropic MLV and contain pseudovirions with mixtures of HIV-1 and ecotropic MLV Env glycoproteins. For this reason, we have begun efforts to produce an uncontaminated, second-generation panel of HeLa-CD4/CCR5 cell clones that express diverse amounts of CCR5 and to isolate a TZM-bl variant lacking MLV antigens.     

Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.     

The FGFRL1 Receptor Is Shed from Cell Membranes, Binds Fibroblast Growth Factors (FGFs), and Antagonizes FGF Signaling in Xenopus Embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.     

Comparison <Emphasis Type="Italic">CCR5del32</Emphasis> Mutation in the <Emphasis Type="Italic">CCR5</Emphasis> Gene Frequencies in Russians,Tuvinians, and in Groups of HIV-Infected Individuals

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 102), Tuvinians (n = 50), and HIV-infected individuals (n = 107) were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.     

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia

The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 10⁹ daltons for mu particle DNA and 0.81 × 10⁹ daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 10⁹ and 5.05 × 10⁹ daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.     

Corin Mutation R539C from Hypertensive Patients Impairs Zymogen Activation and Generates an Inactive Alternative Ectodomain Fragment

Corin is a cardiac transmembrane serine protease that regulates blood pressure by activating natriuretic peptides. Corin variants have been associated with African Americans with hypertension and heart disease. Here, we report a new mutation in exon 12 of the CORIN gene identified in a family of patients with hypertension. The mutation resulted in R539C substitution in the Fz2 (Frizzled-2) domain of the corin propeptide region. We expressed and characterized the corin R539C mutant in HEK293 cells. As determined by Western blot analysis, the R539C mutation did not alter corin expression in transfected cells but impaired corin zymogen activation. In a pro-atrial natriuretic peptide processing assay, the corin mutant had reduced activity and exhibited a dominant-negative effect on wild-type corin. In addition, the R539C mutation altered corin ectodomain shedding, producing an alternative ∼75-kDa fragment that was biologically inactive. Using protease inhibitors and the catalytically inactive corin mutant S985A, we showed that the ∼75-kDa fragment was generated by corin autocleavage. We constructed a series of mutants by replacing single or double Arg residues in the corin propeptide and identified Arg-530 in the Fz2 domain as the alternative autocleavage site. Our results show that the corin mutation R539C identified in hypertensive patients impairs corin zymogen activation and causes an alternative autocleavage that reduces corin activity. These data support that human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension.     

HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex

Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.     

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env''s fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag''s ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

Gene Related to Anergy in Lymphocytes (GRAIL) Expression in CD4+ T Cells Impairs Actin Cytoskeletal Organization during T Cell/Antigen-presenting Cell Interactions

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.     

DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.