Benjamin J. Doranz

A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors

Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.

Epitope Mapping of CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1β binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Regions in β-Chemokine Receptors CCR5 and CCR2b That Determine HIV-1 Cofactor Specificity

Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.

Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells

Significance Dengue virus is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans, affecting over 100 million individuals each year. Currently there is no licensed vaccine or effective antiviral therapy available, and treatment is largely supportive in nature. This study presents a comprehensive analysis of functional T-cell memory against dengue viruses and suggests an HLA-linked protective role for CD8+ T cells. This demonstration of the protective role of T-cell responses points the way forward to identifying robust correlates of protection in natural immunity and vaccination against dengue virus. The role of CD8+ T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8+ responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8+ T cells is associated with protection from dengue virus disease.

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Safe use of the CXCR4 inhibitor ALX40-4C in humans

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.

Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer–dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal–fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.

The Core Domain of Chemokines Binds CCR5 Extracellular Domains while Their Amino Terminus Interacts with the Transmembrane Helix Bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1α and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1α binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1α N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1α by 10–100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1α. In contrast, LD78β, a natural MIP-1α variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1α and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.

Extracellular Cysteines of CCR5 Are Required for Chemokine Binding, but Dispensable for HIV-1 Coreceptor Activity

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40–70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 °C. All cysteine mutants were unable to bind detectable levels of MIP-1β, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.

Genotypic analysis of respiratory mucous sulfation defects in cystic fibrosis.

Intracellular dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to alter endosomal acidification. The most widely studied consequence of this defect has been alterations in the biochemical properties of cystic fibrosis (CF) respiratory mucus glycoproteins. However, studies confirming the existence of mucous processing defects in CF have been hindered by the lack of in vivo animal models by which to test these hypotheses in the absence of secondary effects of chronic bacterial infection. The human bronchial xenograft model has been useful in evaluating the pathophysiologic differences between CF and non-CF airway epithelium, in the absence of secondary disease effects such as goblet cell hyperplasia. In this study we sought to compare the extent of sulfation within secreted mucus glycoproteins from CF and non-CF human bronchial xenografts. Cumulative results of xenografts generated from 13 independent CF tissue samples demonstrated a statistically significant higher level of sulfation (1.7 +/- 0.18, P < 0.026) as compared to non-CF paired controls. Such findings add to the growing body of knowledge that primary defects in sulfation exist in CF respiratory mucin. Correlation of genotype with the extent of mucus sulfation revealed two categories of CF tissues with statistically different mucus sulfation profiles. Results from these studies demonstrated a 2.0 +/- 0.15-fold higher level of mucus sulfation produced from xenografts of five defined CF genotypes as compared to non-CF controls (P < 0.004, n= 10). Interestingly, three CF samples for which one mutant allele remained undefined (deltaoff8/unknown or G551D/unknown) demonstrated no statistical difference in the level of sulfation as compared with matched non-CF controls (n= 3). This as yet unknown allele was not identified within a screen for the 26 most common CF mutations. These results provide preliminary evidence for allelic variation within the CF population which may begin to elucidate the structure-function of CFTR with regards to intracellular mucus processing defects.