Publications by authors named "John C Kappes"

100 Publications

Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein.

bioRxiv 2021 Apr 1. Epub 2021 Apr 1.

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.04.01.438120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020978PMC
April 2021

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects.

J Virol 2020 Dec 11. Epub 2020 Dec 11.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02304-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092844PMC
December 2020

Dual Pathways of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trafficking Modulate the Selective Exclusion of Uncleaved Oligomers from Virions.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail, and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: (i) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that cross-linked efficiently, and was recognized by broadly neutralizing antibodies; (ii) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and (iii) the larger population of uncleaved Env lacked complex carbohydrates, cross-linked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more "open" Env oligomers reached the cell surface in the presence of brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses toward conformationally flexible, uncleaved Env without compromising virus infectivity. The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus type 1 (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this nonfunctional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01369-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925103PMC
January 2021

Long-Acting BMS-378806 Analogues Stabilize the State-1 Conformation of the Human Immunodeficiency Virus Type 1 Envelope Glycoproteins.

J Virol 2020 05 4;94(10). Epub 2020 May 4.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system. The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00148-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199401PMC
May 2020

Antibody-Dependent Cellular Cytotoxicity (ADCC)-Mediating Antibodies Constrain Neutralizing Antibody Escape Pathway.

Front Immunol 2019 11;10:2875. Epub 2019 Dec 11.

Division of Medical Virology, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa.

Both neutralization and antibody-dependent cellular cytotoxicity (ADCC) may be required for effective protection against HIV-1 infection. While there is extensive information on the targets of early neutralizing antibody (nAb) responses, much less is known about the targets of ADCC responses, which are more difficult to characterize. In four individuals recruited during acute HIV-infection, ADCC responses were detected 3-7 weeks prior to nAb responses. To determine the relative influence of ADCC and nAb responses on virus evolution, we performed an in-depth investigation of one individual (CAP63) who showed the highest nAb and ADCC responses. Both nAbs and ADCC antibodies targeted the V4 region of the Env, although there were some differences in epitope recognition. We identified accelerated viral evolution in this region concurrent with emergence of nAb activity, but not ADCC activity. Deep sequencing demonstrated that most nAb escape mutations were strongly selected for, however one nAb escape mutation that rendered the virus highly susceptible to autologous ADCC responses, was suppressed despite not affecting viral fitness. This escape mutation also rendered the virus more sensitive to autologous responses, as well as monoclonal antibodies targeting CD4-induced epitopes, compared to the wildtype virus. In conclusion, ADCC responses and nAbs in donor CAP63 recognized overlapping but unique epitopes in the V4 region, and while ADCC activity was present prior to nAbs, it did not drive viral evolution during this time. However, ADCC responses may select against nAb escape pathways that expose other common ADCC epitopes thereby restricting viral replication and expansion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919271PMC
October 2020

Sexually transmitted founder HIV-1 viruses are relatively resistant to Langerhans cell-mediated restriction.

PLoS One 2019 19;14(12):e0226651. Epub 2019 Dec 19.

Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands.

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922402PMC
April 2020

Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model.

Sci Transl Med 2019 08;11(504)

Lentigen, a Miltenyi Biotec Company, Gaithersburg, MD 20878, USA.

Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4 T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aav5685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136029PMC
August 2019

Primary HIV-1 Strains Use Nef To Downmodulate HLA-E Surface Expression.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany

Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surfaces of infected cells. Recent evidence identified HLA-E, a nonclassical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8 T-cell and natural killer (NK) cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1-infected cells. Here, we examined HLA-E surface levels following infection of CD4 T cells with primary HIV-1 strains and observed that a subset downregulated HLA-E. Two primary strains of HIV-1 that induced the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in a T-cell line, as well as tail swap experiments exchanging the cytoplasmic tail of HLA-A2 with that of HLA-E, demonstrated that Nef modulated HLA-E surface levels and targeted the cytoplasmic tail of HLA-E. Furthermore, infection of primary CD4 T cells with HIV-1 mutants showed that a lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, the results of this study demonstrate for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4 T cells, potentially rendering them less vulnerable to CD8 T-cell recognition but at increased risk of NKG2A NK cell killing. For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic-T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that HIV-1 infection thereby maintained inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell line-adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as an important modulator of CD8 T-cell and NKG2A NK cell functions, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00719-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798123PMC
October 2019

Growth hormone (GH) receptor (GHR)-specific inhibition of GH-Induced signaling by soluble IGF-1 receptor (sol IGF-1R).

Mol Cell Endocrinol 2019 07 14;492:110445. Epub 2019 May 14.

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, AL, 35294, USA; Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA; Endocrinology Section, Medical Service, Veterans Affairs Medical Center, Birmingham, AL, 35233, USA. Electronic address:

Human growth hormone (GH) binds and activates GH receptor (GHR) and prolactin (PRL) receptor (PRLR). LNCaP human prostate cancer cells express only GHR. A soluble fragment of IGF-1 receptor (IGF-1R) extracellular domain (sol IGF-1R) interacts with GHR and blocks GH signaling. We now explore sol IGF-1R's specificity for inhibiting GH signaling via GHR vs. PRLR and test GHR and PRLR extracellular domain inhibition determinants. Although T47D human breast cancer cells express GHR and PRLR, GH signaling is largely PRLR-mediated. In T47D, sol IGF-1R inhibited neither GH- nor PRL-induced STAT5 activation. However, sol IGF-1R inhibited GH-induced STAT5 activation in T47D-shPRLR cells, which harbor reduced PRLR. In MIN6 mouse β-cells, bovine GH (bGH) activates mouse GHR, not PRLR, while human GH activates mouse GHR and PRLR. In MIN6, sol IGF-1R inhibited bGH-induced STAT5 activation, but partially inhibited human GH-induced STAT5 activation. These findings suggest sol IGF-1R's inhibition is GHR-specific. Using a cellular reconstitution system, we compared effects of sol IGF-1R on signaling through GHR, PRLR, or chimeras in which extracellular subdomains 2 (S2) of the receptors were swapped. Sol IGF-1R inhibited GH-induced STAT5 activation in GHR-expressing, not PRLR-expressing cells, consistent with GHR specificity of sol IGF-1R. Interestingly, we found that GHR S2 (which harbors the GHR-GHR dimer interface) was required, but not sufficient for sol IGF-1R inhibition of GHR signaling. These results suggest sol IGF-1R specifically inhibits GH-induced GHR-mediated signaling, possibly through interaction with GHR S1 and S2 domains. Our findings have implications for GH antagonist development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2019.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613819PMC
July 2019

Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses.

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

Centre de Recherche du CHUM, Montreal, QC, Canada

HIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., the luciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4 T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4 T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC. In-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00484-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002730PMC
July 2018

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

Biochim Biophys Acta Biomembr 2018 May 7;1860(5):1193-1204. Epub 2018 Feb 7.

Department of Cell Biology and Biochemistry, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA. Electronic address:

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric T > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2018.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319260PMC
May 2018

Potent NK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein.

J Virol 2017 10 27;91(20). Epub 2017 Sep 27.

Departments of Microbiology and Immunology and Pediatrics, Albert Einstein College of Medicine, Bronx, New York, USA

Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00937-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625480PMC
October 2017

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.

PLoS Pathog 2017 02 27;13(2):e1006226. Epub 2017 Feb 27.

Department of Medicine, University of Colorado School of Medicine, Aurora, CO, United States of America.

Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF) HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a molecular blueprint to gain basic insights in mucosal HIV-1 pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344538PMC
February 2017

Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein.

Virol J 2017 02 16;14(1):33. Epub 2017 Feb 16.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, 450 Brookline Avenue, CLS 1010, Boston, MA, 02215, USA.

Background: The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env), a Type 1 transmembrane protein, assembles into a trimeric spike complex that mediates virus entry into host cells. The high potential energy of the metastable, unliganded Env trimer is maintained by multiple non-covalent contacts among the gp120 exterior and gp41 transmembrane Env subunits. Structural studies suggest that the gp41 transmembrane region forms a left-handed coiled coil that contributes to the Env trimer interprotomer contacts. Here we evaluate the contribution of the gp41 transmembrane region to the folding and stability of Env trimers.

Methods: Multiple polar/charged amino acid residues, which hypothetically disrupt the stop-transfer signal, were introduced in the proposed lipid-interactive face of the transmembrane coiled coil, allowing release of soluble cleavage-negative Envs containing the modified transmembrane region (TM). We also examined effects of cleavage, the cytoplasmic tail and a C-terminal fibritin trimerization (FT) motif on oligomerization, antigenicity and functionality of soluble and membrane-bound Envs.

Results: The introduction of polar/charged amino acids into the transmembrane region resulted in the secretion of soluble Envs from the cell. However, these TM Envs primarily formed dimers. By contrast, control cleavage-negative sgp140 Envs lacking the transmembrane region formed soluble trimers, dimers and monomers. TM and sgp140 trimers were stabilized by the addition of a C-terminal FT sequence, but still exhibited carbohydrate and antigenic signatures of a flexible ectodomain structure. On the other hand, detergent-solubilized cleaved and uncleaved Envs isolated from the membranes of expressing cells exhibited "tighter" ectodomain structures, based on carbohydrate modifications. These trimers were found to be unstable in detergent solutions, but could be stabilized by the addition of a C-terminal FT moiety. The C-terminal FT domain decreased Env cleavage and syncytium-forming ability by approximately three-fold; alteration of the FT trimerization interface restored Env cleavage and syncytium formation to near-wild-type levels.

Conclusion: The modified transmembrane region was not conducive to trimerization of soluble Envs. However, for HIV-1 Env ectodomains that are minimally modified, membrane-anchored Envs exhibit the most native structures and can be stabilized by appropriately positioned FT domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12985-017-0704-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314615PMC
February 2017

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers.

J Virol 2017 05 13;91(9). Epub 2017 Apr 13.

Department of Chemistry, University of Kansas, Lawrence, Kansas, USA

HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells. A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02428-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391476PMC
May 2017

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

PLoS One 2017 2;12(2):e0170672. Epub 2017 Feb 2.

Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Department of Microbiology & Immunobiology, Harvard Medical School, Boston, MA, United States of America.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170672PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289478PMC
September 2017

Diversity of Antiviral IgG Effector Activities Observed in HIV-Infected and Vaccinated Subjects.

J Immunol 2016 12 9;197(12):4603-4612. Epub 2016 Nov 9.

Thayer School of Engineering, Dartmouth College, Hanover, NH 03755

Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIV Ab effector activities in polyclonal serum samples from HIV-infected donors, VAX004 vaccine recipients, and healthy HIV-negative subjects using a variety of primary and cell line-based assays, including Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cell-mediated viral inhibition, and Ab-dependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects, and they point to the potential importance of polyfunctional Ab responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1601197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137799PMC
December 2016

Specific stabilization of CFTR by phosphatidylserine.

Biochim Biophys Acta Biomembr 2017 Feb 30;1859(2):289-293. Epub 2016 Nov 30.

Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA. Electronic address:

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7) is a plasma membrane chloride ion channel in the ABC transporter superfamily. CFTR is a key target for cystic fibrosis drug development, and its structural elucidation would advance those efforts. However, the limited in vivo and in vitro stability of the protein, particularly its nucleotide binding domains, has made structural studies challenging. Here we demonstrate that phosphatidylserine uniquely stimulates and thermally stabilizes the ATP hydrolysis function of purified human CFTR. Among several lipids tested, the greatest stabilization was observed with brain phosphatidylserine, which shifted the Tm for ATPase activity from 22.7±0.8°C to 35.0±0.2°C in wild-type CFTR, and from 26.6±0.7°C to 42.1±0.2°C in a more stable mutant CFTR having deleted regulatory insertion and S492P/A534P/I539T mutations. When ATPase activity was measured at 37°C in the presence of brain phosphatidylserine, Vmax for wild-type CFTR was 240±60nmol/min/mg, a rate higher than previously reported and consistent with rates for other purified ABC transporters. The significant thermal stabilization of CFTR by phosphatidylserine may be advantageous in future structural and biophysical studies of CFTR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2016.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237360PMC
February 2017

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke.

Am J Respir Cell Mol Biol 2017 01;56(1):99-108

Departments of 1 Medicine.

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-μm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1165/rcmb.2016-0226OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5248967PMC
January 2017

Therapeutic intervention of proanthocyanidins on the migration capacity of melanoma cells is mediated through PGE2 receptors and β-catenin signaling molecules.

Am J Cancer Res 2015 15;5(11):3325-38. Epub 2015 Oct 15.

Department of Dermatology, University of Alabama at BirminghamBirmingham, AL 35294, USA; Department of Comprehensive Cancer Center, University of Alabama at BirminghamBirmingham, AL 35294, USA; Birmingham Veterans Affairs Medical CenterBirmingham, AL 35233, USA.

Melanoma is a highly aggressive form of skin cancer and a leading cause of death from skin diseases mainly due to its propensity to metastasis. Due to metastatic tendency, melanoma is often associated with activation of Wnt/β-catenin signaling mechanism. Blocking β-catenin activation may be a good strategy to block melanoma-associated mortality. We have shown earlier that grape seed proanthocyanidins (GSPs) inhibit melanoma cell migration via targeting cyclooxygenase-2 (COX-2) overexpression. Here we explored further whether inhibition of inflammatory mediators-mediated activation of β-catenin by GSPs is associated with the inhibition of melanoma cell migration. Our study revealed that PGE2 receptors (EP2 and EP4) agonists promote melanoma cell migration while PGE2 receptor antagonist suppressed the migration capacity of melanoma cells. GSPs treatment inhibit butaprost (EP2 agonist) or Cay10580 (EP4 agonist) induced migration of melanoma cells. Western blot analysis revealed that GSPs reduced cellular accumulation of β-catenin, and decreased the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and MITF, downstream targets of β-catenin in melanoma cells. GSPs also reduced the protein expressions of PI3K and p-Akt in the same set of experiment. To verify that β-catenin is a specific molecular target of GSPs, we compared the effect of GSPs on cell migration of β-catenin-activated (Mel1241) and β-catenin-inactivated (Mel1011) melanoma cells. GSPs inhibit cell migration of Mel1241 cells but not of Mel1011 cells. Additionally, in vivo bioluminescence imaging data indicate that dietary administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet inhibited the migration/extravasation of intravenously injected melanoma cells in lungs of immune-compromised nude mice, and that this effect of GSPs was associated with an inhibitory effect on the activation of β-catenin and its downstream targets, such as MMPs, in lungs as a target organ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697680PMC
January 2016

Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells.

Oncotarget 2016 Feb;7(7):7899-912

Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA.

Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22(phox) and p47(phox)) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47(phox) protein and decreased levels of the membrane-bound p22(phox) protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.6860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884962PMC
February 2016

High-Throughput Humanized Mouse Models for Evaluation of HIV-1 Therapeutics and Pathogenesis.

Methods Mol Biol 2016 ;1354:221-35

Departments of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.

Mice cannot be used as a model to evaluate HIV-1 therapeutics because they do not become infected by HIV-1 due to structural differences between several human and mouse proteins required for HIV-1 replication. This has limited their use for in vivo assessment of anti-HIV-1 therapeutics and the mechanism by which cofactors, such as illicit drug use accelerate HIV-1 replication and disease course in substance abusers. Here, we describe the development and application of two in vivo humanized mouse models that are highly sensitive and useful models for the in vivo evaluation of candidate anti-HIV therapeutics. The first model, hu-spl-PBMC-NSG mice, uses NOD-SCID IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMC) which develop productive splenic HIV-1 infection after intrasplenic inoculation with a replication-competent HIV-1 expressing Renilla reniformis luciferase (HIV-LucR) and enables investigators to use bioluminescence to visualize and quantitate the temporal effects of therapeutics on HIV-1 infection. The second model, hCD4/R5/cT1 mice, consists of transgenic mice carrying human CD4, CCR5 and cyclin T1 genes, which enables murine CD4-expressing cells to support HIV-1 entry, Tat-mediated LTR transcription and consequently develop productive infection. The hCD4/R5/cT1 mice develop disseminated infection of tissues including the spleen, small intestine, lymph nodes and lungs after intravenous injection with HIV-1-LucR. Because these mice can be infected with HIV-LucR expressing transmitted/founder and clade A/E and C Envs, these mouse models can also be used to evaluate the in vivo efficacy of broadly neutralizing antibodies and antibodies induced by candidate HIV-1 vaccines. Furthermore, because hCD4/R5/cT1 mice can be infected by vaginal inoculation with replication-competent HIV-1 expressing NanoLuc (HIV-nLucR)-, this mouse model can be used to evaluate the mechanisms by which substance abuse and other factors enhance mucosal transmission of HIV-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-3046-3_15DOI Listing
October 2016

Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma.

Oncotarget 2016 Jan;7(2):1227-41

Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Melanoma is the most aggressive and deadly form of cutaneous neoplasm due to its propensity to metastasize. Oncogenic BRAF drives sustained activation of the BRAF/MEK/ERK (MAPK) pathway and cooperates with PI3K/AKT/mTOR (PI3K) signaling to induce epithelial to mesenchymal transition (EMT), leading to cell invasion and metastasis. Therefore, targeting these pathways is a promising preventive/therapeutic strategy. We have shown that fisetin, a flavonoid, reduces human melanoma cell invasion by inhibiting EMT. In addition, fisetin inhibited melanoma cell proliferation and tumor growth by downregulating the PI3K pathway. In this investigation, we aimed to determine whether fisetin can potentiate the anti-invasive and anti-metastatic effects of sorafenib in BRAF-mutated melanoma. We found that combination treatment (fisetin + sorafenib) more effectively reduced the migration and invasion of BRAF-mutated melanoma cells both in vitro and in raft cultures compared to individual agents. Combination treatment also effectively inhibited EMT as observed by a decrease in N-cadherin, vimentin and fibronectin and an increase in E-cadherin both in vitro and in xenograft tumors. Furthermore, combination therapy effectively inhibited Snail1, Twist1, Slug and ZEB1 protein expression compared to monotherapy. The expression of MMP-2 and MMP-9 in xenograft tumors was further reduced in combination treatment compared to individual agents. Bioluminescent imaging of athymic mice, intravenously injected with stably transfected CMV-luciferase-ires-puromycin.T2A.EGFP-tagged A375 melanoma cells, demonstrated fewer lung metastases following combination treatment versus monotherapy. Our findings demonstrate that fisetin potentiates the anti-invasive and anti-metastatic effects of sorafenib. Our data suggest that fisetin may be a worthy adjuvant chemotherapy for the management of melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.6237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811456PMC
January 2016

Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.

Protein Expr Purif 2016 Jan 21;117:67-75. Epub 2015 Sep 21.

Dept. of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address:

The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αβγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2015.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684441PMC
January 2016

The Vaginal Acquisition and Dissemination of HIV-1 Infection in a Novel Transgenic Mouse Model Is Facilitated by Coinfection with Herpes Simplex Virus 2 and Is Inhibited by Microbicide Treatment.

J Virol 2015 Sep 8;89(18):9559-70. Epub 2015 Jul 8.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York, USA

Unlabelled: Epidemiological studies have demonstrated that herpes simplex virus 2 (HSV-2) infection significantly increases the risk of HIV-1 acquisition, thereby contributing to the expanding HIV-1 epidemic. To investigate whether HSV-2 infection directly facilitates mucosal HIV-1 acquisition, we used our transgenic hCD4/R5/cT1 mouse model which circumvents major entry and transcription blocks preventing murine HIV-1 infection by targeting transgenic expression of human CD4, CCR5, and cyclin T1 genes to CD4(+) T cells and myeloid-committed cells. Productive infection of mucosal leukocytes, predominantly CD4(+) T cells, was detected in all hCD4/R5/cT1 mice intravaginally challenged with an HIV-1 infectious molecular clone, HIV-Du151.2env-NLuc, which expresses an env gene (C.Du151.2) cloned from an acute heterosexually infected woman and a NanoLuc luciferase reporter gene. Lower genital tract HIV-1 infection after HIV-Du151.2env-NLuc intravaginal challenge was increased ~4-fold in hCD4/R5/cT1 mice coinfected with HSV-2. Furthermore, HIV-1 dissemination to draining lymph nodes was detected only in HSV-2-coinfected mice. HSV-2 infection stimulated local infiltration and activation of CD4(+) T cells and dendritic cells, likely contributing to the enhanced HIV-1 infection and dissemination in HSV-2-coinfected mice. We then used this model to demonstrate that a novel gel containing tenofovir disoproxil fumarate (TDF), the more potent prodrug of tenofovir (TFV), but not the TFV microbicide gel utilized in the recent CAPRISA 004, VOICE (Vaginal and Oral Interventions to Control the Epidemic), and FACTS 001 clinical trials, was effective as preexposure prophylaxis (PrEP) to completely prevent vaginal HIV-1 infection in almost half of HSV-2-coinfected mice. These results also support utilization of hCD4/R5/cT1 mice as a highly reproducible immunocompetent preclinical model to evaluate HIV-1 acquisition across the female genital tract.

Importance: Multiple epidemiological studies have reported that genital herpes simplex virus 2 (HSV-2) infection increases the risk of HIV-1 sexual acquisition by severalfold. Understanding the underlying mechanisms by which HSV-2 facilitates HIV-1 infection and optimizing the efficacy of therapies to inhibit HIV-1 infection during HSV-2 coinfection should contribute to reducing HIV-1 transmission. Using our novel transgenic hCD4/R5/cT1 mouse model infectible with HIV-1, we demonstrated that HSV-2 infection enhances vaginal transmission and dissemination of HIV-1 infection while stimulating recruitment and activation of CD4(+) T cells and dendritic cells in the lower genital tract. HIV acquisition by hCD4/R5/cT1 mice vaginally coinfected with HSV-2 could be completely prevented in almost half the mice by preexposure prophylaxis (PrEP) with a novel gel containing tenofovir disoproxil fumarate (TDF), the tenofovir prodrug, but not with the tenofovir microbicide gel utilized in CAPRISA-004, VOICE, and FACTS-001 clinical trials. The hCD4/R5/cT1 mice represent a new preclinical mouse model to evaluate vaginal HIV-1 acquisition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01326-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542360PMC
September 2015

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

AIDS Res Hum Retroviruses 2015 Dec 23;31(12):1278-96. Epub 2015 Jun 23.

1 Department of Medicine, University of Alabama at Birmingham , Birmingham, Alabama.

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/aid.2015.0074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663642PMC
December 2015

Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140.

J Virol 2015 Aug 27;89(16):8245-57. Epub 2015 May 27.

Department of Chemistry, University of Kansas, Lawrence, Kansas, USA

Unlabelled: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140.

Importance: A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00628-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524223PMC
August 2015

In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice.

J Virol 2015 Jun 1;89(12):6264-74. Epub 2015 Apr 1.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York, USA

Unlabelled: Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection.

Importance: Epidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00563-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474292PMC
June 2015

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Mol Biotechnol 2015 May;57(5):391-405

Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12033-014-9830-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405497PMC
May 2015
-->