Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.     

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.     

Characterization of DNA-protein cross-links formed by treatment of L1210 cells and nuclei with bis(2-chloroethyl)methylamine (nitrogen mustard)

Proteins cross-linked to DNA after nitrogen mustard (HN2) treatment of cells or isolated nuclei were purified in CsCl gradients. The protein-DNA cross-links could be cleaved by incubation in dilute acid and could be stabilized by alkali pretreatment. These results indicate that proteins cross-linked to DNA by HN2 are bound to alkylated purines. Analysis of the DNA-bound proteins on NaDodSO4-polyacrylamide gels showed that primarily large nonhistone proteins are cross-linked to DNA in cells treated with HN2. Very little if any histone is cross-linked to the DNA. Comparison of DNA bound proteins from HN2-treated cells and HN2-treated nuclei showed that in general the same proteins are linked to DNA in both cases, but some qualitative and quantitative differences exist.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.     

Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes

Receptor binding of paramyxovirus attachment proteins and the interactions between attachment and fusion (F) proteins are thought to be central to activation of the F protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F protein interactions and HN protein attachment to sialic acid receptors, HN and F protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN protein receptor binding, cells transfected with HN and F protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F protein-expressing cells resulted in a twofold decrease in amounts of HN and F protein complexes. In contrast, high levels of complexes containing HN protein and an uncleaved F protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F protein cleavage are necessary for disassociation of the HN and F protein-containing complexes.     

Human Parainfluenza Virus Infection of the Airway Epithelium: Viral Hemagglutinin-Neuraminidase Regulates Fusion Protein Activation and Modulates Infectivity

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN′s receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN′s F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN′s F-triggering activity may have promise in vivo.Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry, the viral surface glycoproteins hemagglutinin-neuraminidase (HN) (the receptor-binding molecule) and F (the fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. To understand these mechanisms, elucidate how paramyxoviruses enter cells, and develop strategies to prevent or treat infection, we study human parainfluenza virus (HPIV), an important cause of croup and bronchiolitis in children. Our results have uncovered fundamental roles of the receptor-binding protein in paramyxovirus fusion and principles of coordinated interaction between the glycoproteins during the viral life cycle.To understand how the diverse functions of the viral glycoproteins are regulated during the viral life cycle, we have used viruses bearing variant HN molecules with mutations at the binding/F-triggering site (and/or the primary receptor-binding site) to study how this molecule works to trigger F (2, 3, 7, 10, 15, 18, 20). The correct timing of F activation (triggering) by HN is essential for entry. For infection to occur, triggering must occur only when F is in proximity to the target cell membrane, and we propose that the regulation of F triggering is essential for the survival of the virus. The outcome of infection is determined by the target cell''s properties and its receptors, and specific mechanisms that are relevant to pathogenesis need to be tested using tissues that reflect the natural host. We therefore tested the hypothesis that a dysregulation of F triggering precludes successful infection in both a cotton rat model and the natural host airway epithelium.For the cotton rat model, previous studies suggested that altered pathogenesis in HPIV infection might be caused by specific HN mutations (24). The present detailed studies of the cotton rat using HN viral variants suggest that the extent of lung infection correlates with the ability of each variant to grow in vivo. The most striking finding is that the ability of the HN variants to grow in vivo is inversely related to their ability to fuse a monolayer of cultured cells. In order to understand the determinants of infection in the natural host, we therefore turned to a model that closely reflects the natural human host tissue, the human airway epithelium (HAE). This model utilizes a recently developed method for culturing primary HAE cells at an air-liquid interface, generating a differentiated, pseudostratified, mucociliary epithelium that faithfully represents the HAE (16). The HAE model was previously used to characterize the polarity and cell specificity of respiratory syncytial virus (26) and HPIV type 3 (HPIV3) (25), confirming that it is suited to studying paramyxovirus-HAE interactions that reflect those in the human lung.We used viruses bearing HNs that are altered in receptor binding or F triggering to reveal the functional relevance of these properties in the HAE and to establish the key role of HN binding site II in infection in the natural host. We propose that an enhanced triggering of F by HN may be a disadvantage in vivo and that the function and timing of F triggering are critical in the target tissue. The correct balance between the three functions of HN (receptor binding, receptor cleaving, and F triggering) resides in the functions of the two binding sites (18), binding and release in site I and F triggering in site II, and both sites of HN play key roles in the natural host.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.     

Physiochemical studies on interactions between DNA and RNA polymerase. Ultraviolet absorption measurements.

The interaction between Escherichia coli RNA polymerase and a restriction fragment of coliphage T7 DNA containing four promoter sites for the coli enzyme has been studied by difference uv absorption spectroscopy in a low ionic strength buffer containing 10 mm MgCl2 and 50 mM KCl. The binding of the enzyme to the DNA is accompanied by a hyperchromic shift which shows a maximum around 260 nm, and increases with increasing temperature in the temperature range studied (4-40 degrees C). Measurements were also carried out with whole T7 DNA and a restriction fragment containing no promoter site. A comparison of the results obtained with the various DNAs suggests that the binding of an RNA polymerase to a promoter site in the low ionic strength medium causes the disruption of a short segment of the DNA helix, of the order of ten pairs; the binding of an enzyme molecule to a promotor site appears to have a cooperative effect on the binding of the enzyme molecules to adjacent non-promoter sites with concomitant disruption of DNA base pairs.     

Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses.

The fusion glycoprotein (F) and hemagglutinin-neuraminidase (HN) genes of human parainfluenza virus type 2 (PI2) were molecularly cloned and expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Expression of the F and HN proteins was detected by using immunoprecipitation and surface immunofluorescence staining. Although the F protein was found to be cleaved into F1 and F2 and expressed on cell surfaces, no cell fusion was observed. However, cotransfection of the F-protein gene together with the P12 HN gene resulted in significant levels of cell fusion. Cell fusion was also observed when separate cell cultures were transfected with the HN and F genes and the F-expressing cells were mixed with the HN-expressing cells. Surprisingly, when the PI2 F protein was expressed together with the parainfluenza virus type 3 (PI3) HN protein, no fusion was detectable in the transfected cells. Similarly, no fusion was found upon coexpression of the PI2 HN and PI3 F proteins. However, coexpression of the PI3 F and HN proteins resulted in extensive cell fusion, which resembled the PI2 coexpression result. These results indicate that under the conditions used, the F protein is unable to cause fusion by itself and the HN protein provides a specific function in cell fusion which cannot be provided by another paramyxovirus attachment protein. Further, the results suggest that a type-specific functional interaction between the F and HN proteins is involved in mediating cell fusion.     

Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cells.

For many paramyxoviruses, including Newcastle disease virus (NDV), syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins. Because a potential interaction in paramyxovirus-infected cells has never been demonstrated, such as interaction was explored by using coimmunoprecipitation and cross-linking. Both HN and F proteins could be precipitated with heterologous antisera after a 5-min radioactive pulse as well as after a 2-h chase in nonradioactive medium, but at low levels. Chemical cross-linking increased detection of complexes containing HN and F proteins at the cell surface. After cross-linking, intermediate- as well as high-molecular-weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after cross-linking resulted in the detection of complexes which electrophresed in the stacker region of the gel, from 160 to 300 kDa, at 150 kDa, and at 74 kDa. Precipitates obtained with anti-F after cross-linking contained species which migrated in the stacker region of the gel, between 160 and 300 kDa, at 120 kDa, and at 66 kDa. The three to four discrete complexes ranging in size from 160 to 300 kDa contained both HN and F proteins when precipitated with either HN or F antisera. That cross-linking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating cross-linking at early time points postinfection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically cross-linked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1 protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the rough endoplasmic reticulum. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a conformational change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion.     

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.     

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.     

The A20R Protein Is a Stoichiometric Component of the Processive Form of Vaccinia Virus DNA Polymerase

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl(2) or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of approximately 48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent approximately 45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA(-) phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308-12318, 2001).     

Comitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways

We have recently developed a mAb, anti-1F7, which defines a family of structures found to include the molecule recognized by anti-Ta1 (CD26). In this paper, we demonstrated that binding of 1F7 by solid-phase immobilized anti-1F7 mAb but not anti-Ta1 mAb has a comitogenic effect by inducing proliferation of human CD4+ T lymphocytes in conjunction with submitogenic doses of anti-CD3 or anti-CD2. The proliferative response induced via the CD3-1F7 or CD2-1F7 pathways is associated with the IL-2 autocrine pathway, including IL-2 production. IL-2R expression and anti-IL-2R (Tac) inhibition. Furthermore, solid-phase immobilization of anti-1F7 but not anti-Ta1 acts in conjunction with submitogenic doses of PMA to mediate a comitogenic effect in the absence of anti-CD3 or anti-CD2, leading to CD4+ T cell proliferation. PMA treatment, in the meantime, leads to enhanced expression of 1F7 on the T cell surface. Despite its functional association with both pathways of activation, however, the 1F7 structure is not comodulated with the CD3/TCR complex nor the CD2 molecule. These findings thus suggest that the CD26 Ag is involved in CD3 and CD2-induced human CD4+ T cell activation.     

Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.     

Association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces.

We previously observed that cell fusion caused by human parainfluenza virus type 2 or type 3 requires the expression of both the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the same virus type, indicating that a type-specific interaction between F and HN is needed for the induction of cell fusion. In the present study we have further investigated the fusion properties of F and HN proteins of parainfluenza virus type 1 (PI1), type 2 (PI2), and type 3 (PI3), Sendai virus (SN), and simian virus 5 (SV5) by expression of their glycoprotein genes in HeLa T4 cells using the vaccinia virus-T7 transient expression system. Consistent with previous results, cell fusion was observed in cells transfected with homotypic F/HN proteins; with one exception, coexpression of any combination of F and HN proteins from different viruses did not result in cell fusion. The only exception was found with the closely related PI1 HN and SN HN glycoproteins, either of which could interact with SN F to induce cell fusion upon coexpression as previously reported. By specific labeling and coprecipitation of proteins expressed on the cell surface, we observed that anti-PI2 HN antiserum coprecipitated PI2 F when the homotypic PI2 F and PI2 HN were coexpressed, but not the F proteins of other paramyxoviruses when heterotypic F genes were coexpressed with PI2 HN, suggesting that the homotypic F and HN proteins are physically associated with each other on cell surfaces. Furthermore, we observed that PI3 F was found to cocap with PI3 HN but not with PI2 HN, also indicating a specific association between the homotypic proteins. These results indicate that the homotypic F and HN glycoproteins are physically associated with each other on the cell surface and suggest that such association is crucial to cell fusion induced by paramyxoviruses.     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

Influence of the human parainfluenza virus 3 attachment protein's neuraminidase activity on its capacity to activate the fusion protein

In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. F activation was assessed by quantitating the irreversible binding of target erythrocytes (RBC) to HN/F-coexpressing cells in the presence of 4-GU-DANA (zanamivir) to release target cells bound only by HN-receptor interactions; the remaining, irreversibly bound target cells are retained via the fusion protein. In cells coexpressing wild-type (wt) or variant HNs with wt F, the fusion promotion capacity of HN was distinguished from target cell binding by measuring changes with time in the amounts of target RBC that were (i) reversibly bound by HN-receptor interaction (released only upon the addition of 4-GU-DANA), (ii) released by HN's neuraminidase, and (iii) irreversibly bound by F-insertion or fusion (F triggered). For wt HN, lowering the pH (to approach the optimum for HPIV3 neuraminidase) decreased F triggering via release of HN from its receptor. An HN variant with increased receptor binding avidity had F-triggering efficiency like that of wt HN at pH 8.0, but this efficiency was not decreased by lowering the pH to 5.7, which suggested that the variant HN's higher receptor binding activity counterbalanced the receptor dissociation promoted by increased neuraminidase activity. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37 degrees C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HN's competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).