Adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue.

Unlabelled: Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F).

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery.

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes.

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.

Fusion promotion by a paramyxovirus hemagglutinin-neuraminidase protein: pH modulation of receptor avidity of binding sites I and II.

Paramyxoviruses, including the childhood respiratory pathogen human parainfluenza virus type 3 (HPIV3), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving (neuraminidase), as well as receptor-binding, activity. HN is a type II transmembrane glycoprotein, present on the surface of the virus as a tetramer composed of two dimers. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane.

Inhibition of hendra virus fusion.

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes.

Paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three different activities: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. These three discrete properties each affect the ability of HN to promote viral fusion and entry. For human parainfluenza type 3, one bifunctional site on HN can carry out both binding and neuraminidase, and the receptor mimic, zanamivir, impairs viral entry by blocking receptor binding.

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.

Inhibition of parainfluenza virus type 3 and Newcastle disease virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites.

Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding.

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.

Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir).

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANA-resistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations.

Human parainfluenza virus type 3 HN-receptor interaction: effect of 4-guanidino-Neu5Ac2en on a neuraminidase-deficient variant.

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity.

A single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity.

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity.

Mechanism of interference mediated by human parainfluenza virus type 3 infection.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection.

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA.

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells.

The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3.

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity.

Enzymic composition and growth rate of human pleural mesothelioma transplants in nude mice.

The enzymic composition of 7 human mesothelioma lines propagated in nude mice was compared with 4 of the original and 15 additional mesotheliomas sampled during the patients' surgery. The xenografts exhibited several-fold higher thymidine kinase (TK), uridine kinase (UK), phosphoserine phosphatase (PSP) and peptidyl proline hydroxylase (PPH) concentrations than the fresh human samples, while their DNA, gamma-glutamyl transpeptidase (GGT) and beta-galactosidase (Bgal) contents remained similar. The volume growth rate of the xenografts (doubling time, DT = 9.

Neutrophil maturation and hypersegmentation promoted in normal bone marrow by a carcinoma-elaborated protein factor.

In rats with subcutaneously transplanted mammary carcinoma 5A (MC) and 2 to 10-fold elevations in the blood content of mature neutrophils, 30-50% of the neutrophils showed pronounced hypersegmentation. This phenomenon could be reproduced in liquid culture of bone marrow cells from normal (tumor-free) animals by 48 hr incubation with the MC host's serum, or with MC-conditioned medium whose activity was attributable to an over 50,000 MW protein. The effects in vitro, occurring without change in total cell number and accompanied by increases in gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) activity, included decreases in the percents of progenitors (myeloblasts, promyelocytes, myelocytes, metamyelocytes and bands) and an increase in mature neutrophils 50% of which exhibited obvious hypersegmentation.

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting.

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes.

Mitogen activation of human lymphocytes from normal and acute lymphocytic leukemia (ALL) subjects.

Lymphocytes from acute lymphocytic leukemia (ALL) subjects were converted by mitogens to blast-like cells whose microscopic appearance and rate of formation was indistinguishable from those in mitogen incubated control lymphocytes. In ALL lymphocytes, however, pokeweed mitogen (PWM) failed to stimulate GGT expression; the mean increase it caused in thymidine kinase (TK) activity and thymidine incorporation was normal, though there were appreciable individual variations. These variations were also apparent with concanavalin A (Con A) but, in most ALL cases, TK and thymidine incorporation rose to much higher levels than in Con-A-treated control lymphocytes.

Phenylalanine uptake in neonatal and infant rat brain.

At the same severe elevations in blood phenylalanine (Phe) levels maintained for 4 h, much higher cerebral Phe concentrations were found in 4-day-old than in 16- or 70-day-old rats. In order to compare this developmental change with 14C-Phe influx mediated by the L transport system, the rapid intracarotid injection method was adapted for use in neonatal rats. The brain uptake index (BUI) thus determined for the first time through the suckling period was significantly higher on the 4th day of age than on the 7th or 24th day, while no significant change occurred during subsequent life.

Enhancement of gamma-glutamyl transpeptidase expression in bone marrow cells by colony stimulating factors.

Studies on blood serum from mammary carcinoma (MC) hosts, which promoted gamma-glutamyl transpeptidase (GGT) expression by normal rat bone marrow cells in liquid culture, were extended to various granulocyte-macrophage colony stimulating factors (CSFs). GGT concentration per cell was found to increase (without change in total cell number) by incubation for 48 h with purified CSF-2 gamma and CSF-1 (but not interleukin-3), with human giant cell elaborated GM-CSF and L-cell conditioned medium, as well as with the 3 MC preparations (host serum, MC extract and MC conditioned medium). GGT-inducing ability (per milligram protein) ranked the 7 preparations in the same order as did their proliferative effect (number of colonies per milligram protein) in the standard mouse bone marrow agar culture system.

Characterization of a mammary carcinoma elaborated factor stimulating gamma-glutamyltranspeptidase expression in bone marrow cells.

The elevation of bone marrow gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) content in rats carrying mammary carcinoma 5A (MC), reproduced in a short-term (48-h) liquid culture of normal bone marrow cells, was found to be attributable to a blood-borne protein factor with an apparent molecular weight of 60,000. Partial purification, based on the extent of stimulation of GGT expression in this culture, increased the specific activity of the host serum from 1.5 to 40 units and that of MC extracts from 6 to 560 units.

Diminished lymphocyte and granulocyte gamma-glutamyltranspeptidase activity in acute lymphocytic leukemia and response to chemotherapy.

gamma-Glutamyltranspeptidase (GGT) activity (per mg protein) in blood lymphoid cells of 27 children with acute lymphoblastic leukemia (ALL) (1.05 +/- 0.15) was significantly below that of controls (2.

Enzyme pathology of human mesotheliomas.

In samples of 16 surgically resected mesotheliomas arising from the pleura of the human lung, 6 enzymes from different metabolic pathways, DNA, and mitotic frequency were quantified. The mesotheliomas, irrespective of cell type or grade, showed lower gamma-glutamyl transpeptidase (GGT) concentration than 36 of the 38 pulmonary adenocarcinomas. The mean concentration of this enzyme in the 15 mesotheliomas was an eighth of that in the 56 carcinomas, whereas their DNA content was similar.