Cells Persistently Infected with Newcastle Disease Virus III. Thermal Stability of Hemagglutinin and Neuraminidase of a Mutant Isolated from Persistently Infected L Cells

Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV(o)). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of Vibrio cholera filtrate neuraminidase; this suggests that the failure of NDV(pi) to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV(o) and NDV(pi) when tested with a soluble substrate, indicating that the inefficient elution of NDV(pi) was not due to a reduced enzyme content. The neuraminidase activity of intact NDV(pi) virions was significantly more stable at 55 C than the enzyme of NDV(o) virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV(pi) may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV(pi) was significantly more resistant to thermal inactivation than that of the wild-type NDV(o). This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.     

Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells

Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [³H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C . perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.     

Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture

The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embryo (CE) cells prepared in different ways to compare efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO₂-incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus than CE cells prepared by our previous standard method.     

Thermal Inactivation of Newcastle Disease Virus I. Coupled Inactivation Rates of Hemagglutinating and Neuraminidase Activities

The thermal stability of Newcastle disease virus has been characterized in terms of the rate constants for inactivation of hemagglutinating activity (HA), neuraminidase activity (NA), and infectivity. Inactivation of HA results in the concomitant loss of NA. Infectivity, however, is much more thermolabile. Disintegration of the virus particle is not responsible for the identical rate constants for inactivation of HA and NA, nor is their parallel inactivation uncoupled in envelope fragments produced by pretreating the virus with phospholipase-C. The data indicate that a common envelope factor(s) can influence the thermal stability of both activities.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)     

Influence of the Rate of Cell Growth and Cell Density on Interferon Action in Chick Embryo Cells

Chick embryo cells became more sensitive to the action of interferon the longer they remained in culture. This phenomenon was found even before confluency had been reached. The relative insensitivity of newly seeded cells was not due to a loss of receptors. Cells synthesizing deoxyribonucleic acid (DNA) at a high rate were less sensitive to interferon action than cells synthesizing DNA at a low rate, but the inhibition of DNA synthesis had no effect on interferon action. An increase in the number of cells used for seeding resulted in an earlier appearance of increased sensitivity to interferon action. These results are discussed in relation to the induction process in animal cells.     

Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.     

Sialyltransferase and Neuraminidase Levels/Ratios and Sialic Acid Levels in Peripheral Blood B Cells Correlate with Measures of Disease Activity in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis: A Pilot Study

Objective

We attempted to determine whether the level of enzymes sialyltransferase (ST) and neuraminidase (Neu) and sialic acid (SIA) in patients with systemic lupus erythematosus (SLE) correlates with the SLE Disease Activity Index (SLEDAI) and in patients with rheumatoid arthritis (RA) correlates with the Disease Activity Score28 (DAS28).

Methods

We examined cell-surface levels of ST6Gal-1, Neu1, ST3Gal-1, Neu3, α-2,6-SIA, and α-2,3-SIA by using fluorescent anti-enzyme antibodies, fluorescent-conjugated Sambucus nigra lectin, and fluorescent-conjugated Maackia amurensis lectin on blood cells in SLE and RA patients and assessed correlations of these levels with SLEDAI and with DAS28. Areas under the curve (AUC) were calculated for different variables against SLEDAI.

Results

The B-cell ST3Gal-1/Neu3 ratio positively correlated with SLEDAI scores (ρ = 0.409 and P = 0.002, statistically significant after Bonferroni’ correction for multiple analyses.). It was supported by the inverse correlation of B-cell Neu3 levels with SLEDAI scores (ρ = −0.264, P = 0.048). The B-cell ST3Gal-1/Neu3 ratio against SLEDAI yielded an AUC of 0.689, which was comparable to that of anti-dsDNA levels at 0.635. In contrast, both ST3Gal-1 and Neu3 levels of RA B cells (r = 0.376, P = 0.013; r = 0.425, P = 0.005, respectively) correlated positively with high disease-activity DAS28 scores.

Conclusion

B-cell ST3Gal-1/Neu3 ratios in SLE and B-cell ST3Gal-1 and Neu3 levels in RA with high disease-activity DAS28 scores correlated with disease activity measures and may be useful in monitoring disease activities.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Studies on Persistent Infections of Tissue Culture VI. Reversible Changes in Newcastle Disease Virus Populations as a Result of Passage in L Cells or Chick Embryos

Populations of the Victoria strain of Newcastle disease virus (NDV), reisolated from persistently infected L-cell cultures and passed twice in the embryonated hen's egg (NDV_L-E-2), were found to differ strikingly from the original, chick embryo-adapted virus (NDV_o). After exposure of L cells to NDV_o at high multiplicities of infection, all cells became abortively infected; they produced only small aggregates of viral antigen and few, if any, infectious virus particles, but they yielded large amounts of interferon. No cytopathic effects (CPE) were noted, and the cultures survived readily as viral carriers. In contrast, NDV_L-E-2 yielded under similar conditions large quantities of viral antigen and infectious virus particles, but no detectable interferon, and the cultures were rapidly destroyed. This change in “virulence” was at least partially reversible by further serial passages of NDV_L-E-2 in chick embryos, as was evident from a consecutive decrease in CPE with a concomitant increasingly rapid recovery of the L-cell cultures, gradually diminishing yields of infectious viral progeny, and the returning of a capacity to induce interferon synthesis. Thus, NDV_L-E-16 resembled NDV_o in many aspects, except for a less striking reduction in its ability to replicate in L cells. Although a selection of viral variants under the given sets of conditions has not been entirely excluded, the establishment of “avirulence” appears to be largely explained by a gradual accumulation of noninfectious, interferon-inducing components in the course of serial passages in the embryonated hen's egg, and the acquisition of “virulence” by a loss of these components. The evidence is as follows. (i) By a step-wise decrease in the dose of virus and restriction of the analyses to the first infectious cycle, a multiplicity of infection was ultimately reached for all “avirulent” populations at which infected cells produced normal yields of infectious viral progeny; i.e., the interferon-inducing components were diluted to noneffective levels. The lowest multiplicity which resulted in a measurable reduction in infectious virus replication was also the last one to induce detectable interferon synthesis. (ii) All viral clones derived from “avirulent” populations behaved like NDV_L-E-2 rather than like the parent viral suspensions, except that some of them elicited small amounts of interferon in L cells. The interferon-inducing components were reduced or lost in the cloning procedures. The nature of the interferon-inducing components has not been established. These components, which were neutralized by rabbit sera against “virulent” NDV_L-E-2 populations, may represent largely inactive or incomplete virus particles; however, the infectious virus-hemagglutinin ratios of “avirulent” populations were mostly of an order similar to those of “virulent” populations. The interferon-inducing components aborted the infectious process in cells simultaneously invaded by infectious virus particles. The implications of these findings are discussed.     

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development,axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.     

不同诱导子对枇杷悬浮细胞生长及熊果酸、齐墩果酸含量的影响

在已建立的稳定枇杷细胞悬浮培养系的试验中,研究加入诱导子水杨酸（SA）、茉莉酸甲酯（MJ）、水解酪蛋白（CH）和酵母提取物（YE）对枇杷悬浮细胞生长及次生代谢产物熊果酸（UA）、齐墩果酸（OA）含量的影响。结果表明,从细胞生长、UA及OA含量三方面综合考虑,以MJ为100.0 mg.L^-1时培养最佳,此时细胞生长、UA及OA含量分别为对照的1.081、2.540及2.590倍。     

Viral and Host Deoxyribonucleic Acid Synthesis in Shope Fibroma Virus-infected Cells as Studied by Means of High-Resolution Autoradiography

Incorporation of (3)H-thymidine by BSC-1 cells infected with Shope fibroma virus was studied by means of high-resolution electron microscopic radioautography. One-hour pulses with the radioactive precursor were given at various times after infection, during a one-step growth cycle of the virus. In the cytoplasm of infected cells, reacted grains occurred over foci of viroplasm; these foci are believed to represent the true sites of viral deoxyribonucleic acid (DNA) replication. Shope fibroma virus DNA synthesis began before 3 hr postinfection, reached a maximum at 8 to 9 hr, and then declined rapidly. It was demonstrated that the decline in (3)H-thymidine uptake is correlated with the onset of viral morphogenesis. In comparison with the noninfected culture, the nuclear labeling, which reflects host DNA metabolism, was slightly reduced by 4 hr postinfection. Inhibition became more marked as infection progressed, and host DNA synthesis was almost completely suppressed in late stages of viral development.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Effect of Newcastle Disease on Serum Copper, Zinc, Cholesterol, and Carotenoid Values in the Chick

Experimentally induced Newcastle disease virus infection of chicks, with a mortality index of 48%, was accompanied by increased concentrations in serum of copper and cholesterol and decreased concentrations of zinc and total carotenoids. These changes distorted, or were superimposed upon, the rhythmic variability in the normal serum concentrations of each of these moieties. Changing values for copper, zinc, and cholesterol became apparent before any overt signs of disease.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.     

Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation,Cell Fusion,and Pathogenicity

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.Newcastle disease virus (NDV) causes a highly contagious respiratory and neurologic disease in chickens, leading to severe economic losses in the poultry industry worldwide (1). NDV is a member of the family Paramyxoviridae and has a nonsegmented, negative-sense RNA genome consisting of six genes (3′-NP-P-M-F-HN-L-5′) (7). Infection of host cells by NDV is accomplished through the interaction of two surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F protein directs the membrane fusion between the viral and cellular membranes, while the HN protein mediates attachment to sialic acid, has neuraminidase activity, and plays a role in fusion promotion (4).The HN protein of NDV is a type II transmembrane glycoprotein and possesses three spatially distinct domains: the ectodomain, transmembrane domain, and cytoplasmic tail. The globular ectodomain contains the sites for receptor binding and neuraminidase activity, and the transmembrane domain anchors to viral envelopes (8). The cytoplasmic tail domain contains 26 highly conserved amino acids whose functions are not well-known. In a plasmid-based expression system, truncation (23 amino acids) of the cytoplasmic tail caused improper orientation of the HN protein in the membrane insertion (13). In other paramyxoviruses, cytoplasmic tails of the HN proteins are known to play crucial roles in virus budding and assembly (10, 12). Our unsuccessful attempt to recover a recombinant NDV (rNDV) with complete deletion of the HN cytoplasmic tail also suggested that the cytoplasmic tail is required for assembly and budding of NDV. Therefore, in this study, we determined the role of amino acid sequences of the cytoplasmic tail in the NDV replication cycle. Since essential regions of the HN cytoplasmic tail for virus replication are unknown, we consecutively deleted the first 6 nucleotides (nt), 12 nt, or 18 nt of the HN cytoplasmic tail in a full-length antigenomic cDNA of NDV intermediate virulent (mesogenic) strain Beaudette C (BC) (6), thus maintaining the “rule of six” for the NDV genome (Fig. ​(Fig.1A).1A). rNDVs were recovered using our standard protocol (6). We recovered rNDVs containing 2-amino-acid deletion and 4-amino-acid deletion of the HN cytoplasmic tail (rBC/HNΔ2 and rBC/HNΔ4, respectively), indicating that only these 4 amino acids are dispensable in generating infectious virions. Since rNDV containing 6-amino-acid deletion of the HN cytoplasmic tail could not be recovered, we wanted to know the role of amino acids at positions 5 and 6 in NDV replications. The serine residue at position 6 is a potential phosphorylation site. Therefore, to determine whether phosphorylation at this site is crucial for recovery of NDV, we additionally generated rNDVs with substitution of alanine and glutamic acid for serine (rBC/HNS6A and rBC/HNS6E, respectively) to confirm its crucial role in the recovery of rNDV.Open in a separate windowFIG. 1.Constructs of recombinant NDVs containing a deletion or point mutation in the N-terminal cytoplasmic tail of the HN protein and replication and fusion index of recovered viruses in infected cells. (A) Consecutively, 6 nt, 12 nt, or 18 nt of mRNA of the HN cytoplasmic tail in a full-length antigenomic cDNA of NDV was deleted. Deletions in the HN cytoplasmic tails are indicated by the large boldface dashes. In addition, serine at position 6 was substituted with alanine and glutamic acid was substituted by changing guanine to cytidine and adenosine, respectively. (B) In vitro replication of the mutant viruses was determined in virus-infected DF-1 cells at an MOI of 0.01. The viral titers were determined by plaque assay. (C) The fusion index was determined in virus-infected Vero cells at an MOI of 0.1. Cells were stained with hematoxylin-eosin, and the fusion index was calculated as a mean number of nuclei per cell. The assay was performed three times.In vitro replication of recovered viruses was determined by plaque assay in virus-infected DF-1 cells at a multiplicity of infection (MOI) of 0.01 (5). All mutant viruses and the parental virus, rBC, grew to similar titers, indicating that alteration of the HN cytoplasmic tails did not affect their in vitro replication (Fig. ​(Fig.1B).1B). Although the rBC/HNΔ4 mutant had grown well up to 24 h postinfection, a reduction of the viral titer was detected thereafter with rapid and extensive induction of syncytia. Therefore, we determined fusion promotion activity of the mutant viruses by quantitating syncytia in virus-infected Vero cells at an MOI of 0.1 at 30 h postinfection (8) and confirmed increased fusion promotion activity of rBC/HNΔ4 followed by rBC/HNS6A compared to that of rBC (Fig. ​(Fig.1C).1C). Similarly, enhanced fusion activity was observed in other cytoplasmic tail-truncated paramyxoviruses, such as simian virus 5 and measles virus (2, 9). It has been postulated that interaction of matrix (M) protein with the cytoplasmic tails of the glycoproteins involves in a fusion-refractory conformation at the early stage of viral maturation (2). Therefore, these altered HN cytoplasmic tails could assist NDV in gaining its cell fusion competence by modulating this fusion-refractory conformation.In general, the levels of the HN protein contents on the surfaces of virus-infected cells and in the virus particles were more affected by point mutation of serine than by truncation of the cytoplasmic tail. We analyzed surface expression of the HN protein on virus-infected DF-1 cells at an MOI of 0.1. At 24 h postinfection, the cells were labeled with a monoclonal antibody against the NDV HN protein followed by anti-Alexa Fluor 488 conjugate, fixed with 4% paraformaldehyde, and analyzed by a fluorescence-activated cell sorter (AriaII; BD Bioscience) with Flowjo program (Tree Star, Inc.) (Fig. ​(Fig.2A).2A). The percentages of cells expressing the HN proteins were 89 (rBC), 78 (rBC/HNΔ2), 71 (rBC/HNΔ4), 64 (rBC/HNS6A), and 53 (rBC/HNS6E). To analyze incorporation of the HN proteins into the viral particles, the parental and mutant viruses harvested from allantoic fluid samples were purified through a 30% sucrose cushion. The viral proteins were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel (Fig. ​(Fig.2B).2B). We first examined whether the mutant viruses incorporated the same levels of other viral proteins. This assay was performed by determining the ratios of the P protein to M protein. We found that similar levels of the P and M proteins were present among the different mutant viruses (Fig. ​(Fig.2B).2B). We then measured the levels of the HN proteins incorporated into the virus particles by determining the ratios of the HN protein to M protein (Fig. ​(Fig.2C).2C). The pattern of incorporation of the HN proteins into the virus particles was similar with their cell surface expression. The HN protein contents of rBC/HNΔ2 and rBC/HNΔ4 were not significantly different from that of the parental virus (P > 0.05), indicating that truncation of the cytoplasmic tail did not impair its incorporation into the viral particles. In contrast, substitution of glutamic acid for serine decreased incorporation of the HN protein into the viral particles, indicating that serine plays an important role in both cell surface expression of the HN protein and its incorporation into the viral particles.Open in a separate windowFIG. 2.Effect of alteration of the HN cytoplasmic tail on incorporation of the HN proteins into viral particles and their surface expression in DF-1 cells. (A) Surface expression of the NDV HN protein in DF-1 cells was analyzed by a fluorescence-activated cell sorter. At 24 h postinfection, DF-1 cells infected with each virus were stained with monoclonal antibody against the HN protein followed by anti-Alexa Fluor 488 conjugate. (B) Ultracentrifuge-purified viruses from infected allantoic fluid samples were separated by electrophoresis, and the gel was then stained with Coomassie brilliant blue. (C) Ratios of HN protein to M-protein levels from the parental virus and the HN cytoplasmic tail mutant viruses were quantified.We further determined the effect of cytoplasmic tail alteration on the pathogenicity of NDV in embryonated eggs and chicks (Table ​(Table1).1). The mean death time (MDT) was determined as the mean time (h) for the minimum lethal dose of virus to kill all the embryos after inoculation of 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs with virus (1). The criteria for classifying the virulence of NDV strains are as follows: virulent strains take <60 h to kill embryos, intermediate virulent strains take 60 to 90 h to kill embryos, and avirulent strains take >90 h to kill embryos. Two mutant viruses (rBC/HNΔ2 and rBC/HNS6E) showed similar values of MDT compared to rBC (59 h). In contrast, the MDTs of rBC/HNΔ4 and rBC/HNS6A were 50 h and 51 h, respectively. Increased pathogenicity of these two mutants was also confirmed by an intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks (1). The scale of the ICPI value in evaluating the virulence of NDV strains is from 0.00 (avirulent strains) to 2.00 (highly virulent NDV strains). The rBC/HNΔ4 virus had the highest ICPI value (1.61), followed by rBC/HNS6A (ICPI value of 1.58), among the parental and mutant viruses, probably due to their enhanced fusion promotion activity. In contrast, rBC/HNS6E had the lowest ICPI value (1.41), which would be associated with decreased HN protein contents detected in the viral particles and virus-infected cells. In our previous study, decreased HN protein contents in virus particles due to complete deletion of 5′ untranslated regions of the HN gene also resulted in attenuation of the virus in chickens (14). Consistently, rBC/HNΔ2 showed biological characteristics and pathogenicity similar to those of the parental virus, suggesting that aspartic acid and arginine are indispensable for the HN cytoplasmic tail of NDV.

TABLE 1.

Pathogenicity of the HN cytoplasmic tail mutant viruses in embryonated eggs and chicks

LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA : I. Effect of Glucose, Acetate, Pyruvate, and Carmine on Intracellular Content and Extracellular Release of Three Acid Hydrolases

Log-phase Tetrahymena were washed and resuspended in a dilute salt solution supplemented with glucose, acetate, pyruvate, or carmine, as desired, and then incubated for 5 h. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed. Extracellular activities were corrected for proteolytic degradation. The three nutritive substrates affected both the amount and pattern of extracellular enzyme release, but carmine had no effect. Intracellular activities declined early in the starvation period, but partially recovered with time, particularly α-glucosidase activity. Acetate reduced the decline in acid phosphatase activity; acetate and glucose enhanced the recovery of α-glucosidase activity; carmine had no effect on intracellular enzyme activities. Protein content changed little and was unaffected by the addition of substrates. Glycogen content increased during incubation; acetate and glucose enhanced the increase.     

Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Mouse Embryo Cells Infected with Three Classes of Polyoma Virus Particles

Polyoma virus particles were purified by equilibrium centrifugation in CsCl. Particles from three regions of the density gradient were examined for infectivity, for their ability to induce expanded pools of deoxyribonucleic acid (DNA) precursors, and for their ability to stimulate the synthesis of DNA. The most infectious population of particles, the virions, having a buoyant density of 1.33 g/ml, gave the greatest stimulation of the DNA-synthesizing apparatus of mouse embryo cells. Empty particles at density 1.29 g/ml had no DNA stimulatory activity. A population of particles of intermediate density, referred to as pseudovirions, was also much less active than virions in stimulating DNA synthesis, and the limited stimulatory activity of the latter fraction may be accounted for by its measured contamination with infective particles.     

Growth of Trypanosoma cruzi in Cultures of Chick Embryo Cells, and Effects of Furazolidone and Tris (p-Aminophenyl)Carbonium Chloride

SYNOPSIS. Monolayers of cells of coverslips were produced by culturing known numbers of trypsinized chick cells in growth medium (solution 199 plus 20% calf serum) at 37 C for 2 days. The fluid was then replaced with maintenance medium (solution 199 plus 5% calf serum) containing various known numbers of T. cruzi and the preparations were incubated at 33 C for 5 days; fresh maintenance medium was substituted on the 2nd or 3rd day. The inocula of parasites were obtained from T. cruzi -cell cultures, supplemented with 2% sterile NNN overlay, or from NNN cultures.
The numbers of extracellular parasites, proportions of infected cells, and percentage distribution of infected cells relative to the number of intracellular leishmanial bodies were determined on days 2 or 3 and 5 of parasite cultivation in many experiments. Analyses of the data gave the following results. Extracellular parasites increased 2- to 14-fold during the first 2 or 3 days, depending upon the source and size of the inocula, and 10- to 20-fold during the last 2 or 3 days. Cell infection continued throughout incubation at daily rates of 1.4-4.5%; 8-22% of the cells became infected during the 5 days of incubation. Intracellular growth was reflected most clearly by increases in the proportion of cells having >10 leishmanial bodies. This increase was about 5% daily during the last 2 or 3 days of incubation.
A useful test procedure for assessing the antiparasitic action and chick embryo cell toxicity of drugs is illustrated by data obtained with furazolidone and tris ( p -aminophenyl)carbonium chloride.