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Judith M White

Publications and source records attributed to Judith M White.

17 recordsLinked to original sources
PubMed2006

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer ("molecular fontanels"), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Animals↗
PubMed2005

Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity.

The fusion peptide of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, we changed several residues proposed to stabilize the kink in this structure and measured fusion. Among these, mutants E11A and W14A expressed hemagglutinins with hemifusion and no fusion activities, and F9A and N12A had no effect on fusion, respectively. Binding enthalpies and free energies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the parent full-length molecules. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type, which points into the membrane. A specific fixed angle boomerang structure is thus required to support membrane fusion.

Cell Line↗
PubMed2005

Selective modulation of integrin-mediated cell migration by distinct ADAM family members.

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.

ADAM Proteins↗
PubMed2005

Receptor-induced conformational changes in the SU subunit of the avian sarcoma/leukosis virus A envelope protein: implications for fusion activation.

The avian sarcoma/leukosis virus (ASLV) is activated for fusion by a two-step mechanism. For ASLV subgroup A (ASLV-A), association with its receptor (Tva) at neutral pH converts virions to a form that can bind target membranes and, in some assays, induce the lipid-mixing stage of fusion. Low pH is necessary to complete the fusion reaction. ASLV-A env (EnvA) exists on the viral surface as a trimer of heterodimers consisting of receptor binding (SU-A) and fusion-mediating (TM-A) subunits. As the receptor binding and fusion-mediating functions reside in separate subunits, we hypothesize that SU-A and TM-A are conformationally coupled. To begin to understand the effect of the binding of a soluble 47-residue domain of the receptor (sTva) on this coupling and the subsequent function of low pH, we prepared recombinant proteins representing full-length SU-A and a nested set of deletion mutant proteins. Full-length SU-A binds sTva with high affinity, but even small deletions at either the N or the C terminus severely impair sTva binding. We have purified the full-length SU-A subunit and characterized its interactions with sTva and the subsequent effect of low pH on the complex. sTva binds SU-A with an apparent KD of 3 pM. Complex formation occludes hydrophobic surfaces and tryptophan residues and leads to a partial loss of alpha-helical structure in SU-A. Low pH does not alter the off rate for the complex, further alter the secondary structure of SU-A, or induce measurable changes in tryptophan environment. The implications of these findings for fusion are discussed.

Avian Leukosis Virus↗
PubMed2004

Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein.

A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37 degrees C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.

Gene Products, env↗
PubMed2004

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate.

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

Amino Acid Sequence↗
PubMed2003

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion.

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.

Animals↗
PubMed2003

Leash in the groove mechanism of membrane fusion.

Formation of helix bundles has been proposed as a general mechanism for viral and cellular membrane fusion reactions. Class I viral fusion proteins, including HIV Env and influenza hemagglutinin (HA), form six-helix bundles in their fusogenic forms. The HIV Env six-helix bundle extends to the membrane proximal end of the protein, where it is poised to pull the fusing membranes together. In contrast, the HA six-helix bundle is located at the membrane distal end of the protein. It is followed by a C-terminal 'leash' that packs into the grooves and extends to the membrane proximal end of the coiled-coil. Here, we describe the ability of C-terminal leash mutants to change conformation and induce fusion. Our data indicate that packing of the C-terminal leash into the grooves of the coiled-coil is necessary for HA to mediate the lipid mixing stage of fusion, and that hydrophobic membrane proximal leash residues secure this interaction. Therefore, HA employs a 'leash in the groove,' rather than a helix-bundle, mechanism of membrane fusion.

Amino Acid Substitution↗
PubMed2003

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3.

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.

ADAM Proteins↗
PubMed2003

ADAMs: modulators of cell-cell and cell-matrix interactions.

ADAMs contain adhesive and metalloprotease domains. As major ectodomain sheddases, they release a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules and receptors. ADAMs can also cleave and remodel components of the extracellular matrix. Hence, ADAMs are emerging as key modulators of cell-cell and cell-matrix interactions. Important questions, including if and how ADAM adhesive domains promote ADAM protease function, are currently being addressed.

ADAM Proteins↗
PubMed2003

The avian retrovirus avian sarcoma/leukosis virus subtype A reaches the lipid mixing stage of fusion at neutral pH.

We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

3T3 Cells↗
PubMed2002

The cysteine-rich domain regulates ADAM protease function in vivo.

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

ADAM Proteins↗
PubMed2002

A single glycosylation site within the receptor-binding domain of the avian sarcoma/leukosis virus glycoprotein is critical for receptor binding.

Retroviral envelope proteins are heavily glycosylated. In some cases, glycosylation has been shown to be important for folding, protein stability, immune evasion, or receptor usage. The receptor-binding subunit (SU or gp85) of the envelope protein (EnvA) of the avian sarcoma/leukosis virus, subtype A (ASLV-A), contains 11 potential N-linked glycosylation sites (NXS/T). To address the importance of N-linked glycosylation for the function of EnvA, we prepared a series of EnvA proteins lacking one or more of these carbohydrate addition sites. Using site-directed mutagenesis, we mutated the S or T in each NXS/T glycosylation sequon to A. We also prepared EnvAs bearing selected double and triple mutations. We examined each mutant EnvA for its ability to be expressed at the cell surface, proteolytically processed into gp85 and gp37, incorporated into MLV pseudotyped virions, and to support infection of cells expressing the ASLV-A receptor, Tva. Eight single mutations were well tolerated, and, in general, EnvA was able to tolerate double mutations of these glycosylation sites. Triple mutations were more variable in their effects. Of the three glycosylation sites important for EnvA function, two are important for folding (EnvA production and processing were severely impaired). For the third, although EnvA processing was impaired, significant amounts of processed EnvA were expressed at the cell surface and incorporated into virions. Nonetheless, this mutant EnvA, EnvADeltaNg10, was unable to support infection. Further examination of EnvADeltaNg10 revealed that it was unable to bind Tva and was severely impaired for binding to a monoclonal antibody which inhibits receptor binding. This work has therefore identified a single N-linked glycosylation site in the SU domain of EnvA that is critical for binding between EnvA and its receptor, Tva.

Amino Acid Sequence↗
PubMed2002

Novel monoclonal antibody directed at the receptor binding site on the avian sarcoma and leukosis virus Env complex.

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.

Animals↗
PubMed2002

New insights into the spring-loaded conformational change of influenza virus hemagglutinin.

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix "spring-loaded" conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only approximately 50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

3T3 Cells↗