BCA2/Rabring7 Targets HIV-1 Gag for Lysosomal Degradation in a Tetherin-Independent Manner

BCA2 (Rabring7, RNF115 or ZNF364) is a RING-finger E3 ubiquitin ligase that was identified as a co-factor in the restriction imposed by tetherin/BST2 on HIV-1. Contrary to the current model, in which BCA2 lacks antiviral activity in the absence of tetherin, we found that BCA2 possesses tetherin-independent antiviral activity. Here we show that the N-terminus of BCA2 physically interacts with the Matrix region of HIV-1 and other retroviral Gag proteins and promotes their ubiquitination, redistribution to endo-lysosomal compartments and, ultimately, lysosomal degradation. The targeted depletion of BCA2 in tetherin-expressing and tetherin-deficient cells results in a significant increase in virus release and replication, indicating that endogenous BCA2 possesses antiviral activity. Therefore, these results indicate that BCA2 functions as an antiviral factor that targets HIV-1 Gag for degradation, impairing virus assembly and release

Oral Delivery of Bioencapsulated Exendin-4 Expressed in Chloroplasts Lowers Blood Glucose Level in Mice and Stimulates Insulin Secretion in Beta-TC6 Cells

Glucagon like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2 min in circulation). GLP-1 analog, Exenatide (Byetta) has a longer half life (3.3–4 hrs) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because diabetic patients take \u3e60,000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)-fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12–24 fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulfide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5,000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but didn’t cause hypoglycemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost

Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors

ABSTRACT Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development. IMPORTANCE: To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development

Vaccine Protection Against Zika Virus from Brazil

Zika virus (ZIKV) is a flavivirus that is responsible for an unprecedented current epidemic in Brazil and the Americas1,2. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans3–8 and mice9–11. The rapid development of a safe and effective ZIKV vaccine is a global health priority1,2, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization of a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a ZIKV outbreak strain from northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice11. We produced DNA vaccines expressing full-length ZIKV pre-membrane and envelope (prM-Env) as well as a series of deletion mutants. The full-length prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV as measured by absence of detectable viremia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and CD4 and CD8 T lymphocyte depletion in vaccinated mice did not abrogate protective efficacy. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans will likely be readily achievable

Expression And Functional Evaluation Of Exendin 4 Fused To Cholera Toxin B Subunit In Tobacco Chloroplast To Treat Type 2 Diabetes

The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in β-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes

BCA2 re-distributes HIV-1 Gag, but not HIV-1 Gag G₂A, to endo-lysosomal compartments.

<p>(A) Distribution of the HIV-1 Gag-G₂A-GFP mutant in HA-BCA2⁻ (top panel) and HA-BCA2⁺ (bottom panels) cells. (B-C) The intracellular localization of native HIV-1 Gag in the presence of HA-BCA2 was determined by staining cells for p24 (green), HA-BCA2 (blue) and either CD63, or LAMP1 (red). The white scale bar corresponds to 10 µm for images with more than one cell, and 7.5 µm for images with single cells.</p

BCA2 leads to the accumulation of Gag proteins within intracellular compartments.

<p>The effects of BCA2 on Gag distribution were investigated by confocal microscopy. 293T cells were co-transfected with constructs coding for HA-BCA2 and HIV-1 Gag-GFP, SIV Gag-GFP or empty vector. Similar assays were performed in the presence of HIV-1 NL4-3 and SIV_mac239 proviral DNA. Cells were stained for HA-BCA2 (red), Gag-GFP or Capsid (p24 or p27) (green) and the nuclei (blue). (A) Cellular distribution of HA-BCA2 in Gag-deficient cells. (B) Cellular distribution of HIV-1 Gag-GFP, SIV Gag-GFP (left panels) and HIV-1 p24 or SIV p27 (right panels) in HA-BCA2-deficient cells. (C) Cellular distribution of HIV-1 Gag-GFP and HIV-1 p24 in cells expressing HA-BCA2. (D) Cellular distribution of SIV Gag-GFP and SIV p27 in HA-BCA2-expressing cells. The white scale bar corresponds to 10 µm for images with more than one cell, and 7.5 µm for images with single cells.</p

BCA2 promotes the lysosomal degradation of HIV-1 and SIV Gag.

<p>To determine if BCA2 promotes the proteasomal or lysosomal degradation of retroviral Gag proteins, virus release assays for HIV-1 and SIV were performed in the presence of proteasomal inhibitors (A-B) and lysosomal inhibitors (C-D). Virus release was measured by HIV-1 p24 or SIV p27 antigen-capture ELISA and expressed as the percentage of maximal virus release in the absence of HA-BCA2. The cell lysates (WCL) and virions derived from these transfections were analyzed by western blot. Clasto: clasto-Lactacystin β-lactone. Chlor: chloroquine. Error bars represent standard deviation of independent experiments.</p

The N-terminus of BCA2 interacts with the Matrix region of Gag.

<p>(A) To investigate if retroviral Gag proteins interact with BCA2, 293T cells were co-transfected with HIV-1, SIV and Mo-MLV Gag constructs and either empty vector or a vector encoding HA-BCA2. Cell lysates were immunoprecipitated with a CA-specific antibody and membranes were probed with an anti-HA antibody. Results obtained from these assays were corroborated independently twice. (B) Schematic representation of the Gag and HA-BCA2 deleted constructs used. Similar to panel A, HIV (C) and SIV (D) Gag deleted mutants were tested for an interaction with HA-BCA2 by co-immunoprecipitation. Likewise, HA-BCA2 deleted mutants were tested for an interaction with HIV (E) and SIV (F) Gag proteins by co-immunoprecipitation. In this case, lysates were immunoprecipitated with an anti-HA antibody and western blot membranes were probed with a GFP-specific antibody. Whole cell lysates (WCL) were set aside to check the input levels of these proteins and β-actin. IP: immunoprecipitation. V: empty vector.</p

Lysine residues in SIV Gag predicted to become ubiquitinated.

<p>The full coding sequence of SIV Gag was analyzed by a Bayesian Discriminant Method algorithm (<a href="http://bdmpub.biocuckoo.org/" target="_blank">http://bdmpub.biocuckoo.org/</a>). The selected amino acids obtained the highest score for this prediction.</p