The Bitter Taste Receptor TAS2R16 Achieves High Specificity and Accommodates Diverse Glycoside Ligands by using a Two-faced Binding Pocket.

Although bitter taste receptors (TAS2Rs) are important for human health, little is known of the determinants of ligand specificity. TAS2Rs such as TAS2R16 help define gustatory perception and dietary preferences that ultimately influence human health and disease. Each TAS2R must accommodate a broad diversity of chemical structures while simultaneously achieving high specificity so that diverse bitter toxins can be detected without all foods tasting bitter.

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.

The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding.

Atomic-level functional model of dengue virus Envelope protein infectivity.

A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding.

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences.

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis.

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases.

Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies.

CD4 binding affinity determines human immunodeficiency virus type 1-induced alpha interferon production in plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-alpha) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-alpha induction.

Focused dampening of antibody response to the immunodominant variable loops by engineered soluble gp140.

Immunization studies with modified gp120 monomers using a hyperglycosylation strategy, in which undesired epitopes are masked by the selective incorporation of N-linked glycans, were described in a previous paper (Selvarajah S, et al., J Virol 2000;79:12148-12163). In this report, we applied the hyperglycosylation strategy to soluble uncleaved gp140 trimers to improve the antigenic and immunogenic profile in the context of a trimeric conformation of the immunogen.

A simian immunodeficiency virus V3 loop mutant that does not efficiently use CCR5 or common alternative coreceptors is moderately attenuated in vivo.

Sexually transmitted HIV-1 strains utilize the chemokine receptor CCR5 for viral entry and inhibitors targeting this coreceptor offer great promise for antiretroviral therapy. They also raise the question, however, whether viral variants exhibiting altered coreceptor interactions and resistance against these antiviral agents might still be pathogenic. In the present study, we analyzed a SIVmac239 envelope (Env) mutant (239DL) containing two mutations in the V3 loop which reduced viral entry via CCR5 by 10- to 20-fold, disrupted utilization of common alternative SIV coreceptors and changed the way Env engaged CCR5.

A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection.

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo.

Comparing antigenicity and immunogenicity of engineered gp120.

We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein.

Characterization of neutralizing antibodies to West Nile virus.

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity.

An infectious West Nile virus that expresses a GFP reporter gene.

West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene.

Determinants within gp120 and gp41 contribute to CD4 independence of SIV Envs.

Entry of simian immunodeficiency virus (SIV) into cells is mediated by binding of the viral envelope (Env) glycoprotein to cellular CD4 and chemokine receptor molecules. Interaction of the Env gp120 subunit with CD4 induces conformational changes that result in exposure of a conserved coreceptor binding site. The chemokine receptor CCR5 is the major coreceptor used for SIV entry.

Retroviral mRNA nuclear export elements regulate protein function and virion assembly.

Rodent cells are notable for their inability to support normal assembly of HIV particles. In this report, we address possible causes for this defect by considering the hypothesis that mRNA-associated events occurring in the nucleus can regulate the activity of their encoded proteins in the cytoplasm. We show that altering the RNA nuclear export element used by HIV gag-pol mRNA from the Rev response element to the constitutive transport element restores both the trafficking of Gag to cellular membranes and efficient HIV assembly in murine cells.

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors.

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4.

Mutations in the C3 region of human and simian immunodeficiency virus envelope have differential effects on viral infectivity, replication, and CD4-dependency.

Residues within the highly conserved C3 region of human and simian immunodeficiency virus (HIV, SIV) envelope proteins (Envs) bind directly to the cellular CD4 receptor. However, substitutions of D385, which is critical for CD4 engagement along with other changes such as G382R, G383R, frequently arise in SIV mac-infected macaques. We investigated the influence of substitutions in the SIVmac and HIV-1 C3 regions on viral entry, dependence on CD4, and replication.

Structure and function of CC-chemokine receptor 5 homologues derived from representative primate species and subspecies of the taxonomic suborders Prosimii and Anthropoidea.

A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.

Unique pattern of convergent envelope evolution in simian immunodeficiency virus-infected rapid progressor macaques: association with CD4-independent usage of CCR5.

The rate of disease development in simian immunodeficiency virus (SIV) infection of macaques varies considerably among individual macaques. While the majority of macaques inoculated with pathogenic SIV develop AIDS within a period of 1 to 2 years, a minority exhibit a rapid disease course characterized by absence or transience of humoral and cellular immune responses and high levels of virus replication with widespread dissemination of SIV in macrophages and multinucleated giant cells. The goal of this study was to examine viral evolution in three SIVsmE543-3-inoculated rapid progressors to determine the contribution of viral evolution to the development of rapid disease and the effect of the absence of immune pressure upon viral evolution.

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity.

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion.

A replication-competent, neutralization-sensitive variant of simian immunodeficiency virus lacking 100 amino acids of envelope.

Coding sequences for the first two variable loops of the gp120 envelope glycoprotein were removed from simian immunodeficiency virus (SIV) strain 239 (SIVmac239). This deletion encompassed 100 amino acids. The resulting virus replicated poorly after transfection into immortalized T-cell lines, with peak replication occurring only after 25 to 30 days.

Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication.

We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L.