Nature of virally mediated changes in membrane permeability to small molecules.

1. The changes in membrane permeability to small molecules caused by Sendai virus [Pasternak & Micklem (1973) J. Membr. Biol. 14, 293-303] have been further characterized. The uptake of substances that are concentrated within cells is inhibited. Choline and 2-deoxyglucose, which become phosphorylated, and aminoisobutyrate and glycine, which are driven by a Na+-linked mechanism, are examples. The uptake of each compound under conditons where its diffusion across the plasma membrane is rate-limiting is stimulated by virus. Choline, 2-deoxyglucose and amino acids at high concentration, amino acids in Na+-free medium, and most substances at low temperature, are examples. It is concluded that virally mediated decrease of uptake is due to one of two causes. Substances that are accumulated by phosphorylation are not retained because of leakage of the phosphorylated metabolites out of cells. Substances that are accumulated by linkage to a Na+ gradient are no longer accumulated because of collapse of the gradient resulting from an increased permeability to Nat 2. Increased permeability to K+ and Na+ results in (a) membrane depolarization and (b) cell swelling. The latter event leads to haemolysis (for erythrocytes) and can lead to giant-cell (polykaryon) formation (for several cell types). 3. Recovery of cells can be temporarily achieved by the addition of Ca2+; permanent recovery requires incubation for some hours at 37 degrees C. 4. The possible significance of virally mediated permeability changes, with regard to clinical situations and to cell biology, is discussed.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.     

Polypeptide linkages and resulting structural features as powerful chromogenic factors in the Lowry phenol reaction. Studies on a glycoprotein containing no Lowry phenol-reactive amino acids and on its desialylated and deglycosylated products.

With bovine serum albumin as the reference standard, the armadillo salivary-gland glycoprotein, although containing no chromogenic amino acids and only small amounts of colour-yielding peptides [Chou & Goldstein (1960) Biochem. J. 75, 100-115], is highly reactive in the Lowry phenol protein assay [Wu & Pigman (1977) Biochem. J. 161, 37-47]. After desialylation and Smith degradation of the glycoprotein, the Lowry phenol value increased by 13 and 30% respectively, which suggests that both sialic acid and N-acetylhexosamine exert shielding effects in this reaction. Acid hydrolysis for 30 min decreased the Lowry phenol value by more than 45%, which indicates that the peptide linkages and steric features affect the Lowry phenol reactivity. After hydrolysis for up to 6h, the remaining Lowry phenol value of the partially hydrolysed core protein paralleled the amount of unhydrolysed peptides, inferring that both acid-sensitive and acid-resistant chromophoric peptides are fairly evenly distributed along the whole polypeptide chain. As with bovine serum albumin, more than 80% of the colour yield obtained in the Lowry phenol assay with this glycoprotein is Cu2+-dependent.     

The hemagglutinins of the human influenza viruses A and B recognize different receptor microdomains

A cryptically I-active sialylglycoprotein (glycoprotein 2) isolated from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y., Suzuki, T. and Matsumoto, M. (1983) J. Biochem. 93, 1621-1633) contains N-glycolylneuraminic acid (NeuGc) as its predominate sialic acid and exhibits poor receptor activity for a variety of influenza viruses. Enzymatic modification of asialoglycoprotein-2 to contain N-acetylneuraminic acid (NeuAc) in the NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal sequences using specific sialyltransferase resulted in the appearance of receptor activity toward human influenza viruses A and B. The biological responsiveness chicken erythrocytes treated with sialidase and then reconstituted with derivatized glycoprotein 2 showed considerable recovery to influenza virus hemagglutinin-mediated agglutination, low-pH fusion and hemolysis. Specific hemagglutination inhibition activity of derivatized glycoprotein 2 was 5-16-times higher than that of human glycophorin. A/PR/8/34 (H1N1) virus preferentially recognized derivatized glycoprotein 2 containing NeuAc alpha 2-3Gal sequence over that containing NeuAc alpha 2-6Gal while the specificity of A/Aichi/2/68 (H3N2) for the sialyl linkages was reversed. B/Lee virus recognized both sequences almost equally. The biological responsiveness to the viruses of the erythrocytes labeled with the derivatized glycoprotein 2 containing NeuGc was considerably lower than that of derivatized glycoprotein 2 containing NeuAc. The results demonstrate that the hemagglutinins of human isolates of influenza viruses A and B differ in the recognition of microdomains (NeuAc, NeuGc) of the receptors for binding and fusion activities in viral penetration and the sequence to which sialic acid (SA) is attached (SA alpha 2-3Gal, SA alpha 2-6Gal). Inner I-active neolacto-series type II sugar chains may be important in revealing the receptor activity toward the hemagglutinin of both human influenza viruses A and B.     

An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid.

Rates of protein synthesis in tissues can be calculated from the specific radioactivity of free and protein-bound amino acids at the end of a constant infusion of a labelled amino acid (Garlick, Millward & James (1973) Biochem. J. 136, 935--945]. The simplifying assumptions used in these calculations have been criticized [Madsen, Everett, Sparrow & Fowkes (1977) FEBS Lett. 79, 313--316]. A more detailed analysis using a programmable desk-top calculator is described, which shows that the errors introduced by the simplifying assumptions are small, particularly when the specific radioactivity of the free amino acid rises rapidly to a constant value.     

New Insights into the Hendra Virus Attachment and Entry Process from Structures of the Virus G Glycoprotein and Its Complex with Ephrin-B2

Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a “latch” to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.     

The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase.

The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction.     

Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates.

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5'-monophosphates. The purine nucleoside 5'-monophosphates are more effective than the pyrimidine homologues. 5'-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825--835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309--316].     

Lassa virus glycoprotein complex review: insights into its unique fusion machinery

Lassa virus (LASV), an arenavirus endemic to West Africa, causes Lassa fever—a lethal hemorrhagic fever. Entry of LASV into the host cell is mediated by the glycoprotein complex (GPC), which is the only protein located on the viral surface and comprises three subunits: glycoprotein 1 (GP1), glycoprotein 2 (GP2), and a stable signal peptide (SSP). The LASV GPC is a class one viral fusion protein, akin to those found in viruses such as human immunodeficiency virus (HIV), influenza, Ebola virus (EBOV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are enveloped and utilize membrane fusion to deliver their genetic material to the host cell. Like other class one fusion proteins, LASV-mediated membrane fusion occurs through an orchestrated sequence of conformational changes in its GPC. The receptor-binding subunit, GP1, first engages with a host cell receptor then undergoes a unique receptor switch upon delivery to the late endosome. The acidic pH and change in receptor result in the dissociation of GP1, exposing the fusion subunit, GP2, such that fusion can occur. These events ultimately lead to the formation of a fusion pore so that the LASV genetic material is released into the host cell. Interestingly, the mature GPC retains its SSP as a third subunit—a feature that is unique to arenaviruses. Additionally, the fusion domain contains two separate fusion peptides, instead of a standard singular fusion peptide. Here, we give a comprehensive review of the LASV GPC components and their unusual features.     

Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity--5'5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody--inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.     

Immunochemical properties of short recombinant polypeptides of proteine of, the West Nile virus bearing epitopes of domains I and II

Fragments cDNA (nt 935-1475, 1091-1310, 935-1193) encoding N-terminal part of protein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human were obtained and cloned. Recombinant polypeptides of glycoprotein E (E1-86, E53-126, E1-180) of the WNV with corresponding amino acid sequence to the cloned fragments of cDNA and modeling the epitopes of domains I and II of surface glycoprotein E were purified by affinity chromatography. Twelve types of monoclonal antibodies (MAbs) created in our laboratory against recombinant polypeptide E1-180 interact with glycoprotein E of the WNV as results of Western blot and ELISA that is demonstrating an similarity of chemical structure of short recombinant polypeptides and corresponding amino acid sequence regions of WNV protein E. Analysis of interactions of MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus let us reveal no less than six epitopes within domains I and II of glycoprotein E of the WNV. No less than seven types of MAbs to 86-126 aa region of the domain II were found where located peptide providing fusion of virus--cell membranes (98-110 aa). The epitope for anti-receptor MAbs 10H10 within 53-86 aa region of domain II of protein E of the WNV was mapped and it shows that the fusion peptide and co-receptor of protein E for cellular laminin-binding protein (LBP) are spatial nearness. X-ray model of protein E let us suppose that bc-loop (73-89 aa) of domain II interacts with LBP and together with cd-loop (fusion peptide) determines an initial stages of penetration virions into cell.     

Importance of experimental conditions in evaluating the malonyl-CoA sensitivity of liver carnitine acyltransferase. Studies with fed and starved rats.

The experiments reconfirm the powerful inhibitory effect of malonyl-CoA on carnitine acyltransferase I and fatty acid oxidation in rat liver mitochondria (Ki 1.5 microM). Sensitivity decreased with starvation (Ki after 18 h starvation 3.0 microM, and after 42 h 5.0 microM). Observations by Cook, Otto & Cornell [Biochem. J. (1980) 192, 955--958] and Ontko & Johns [Biochem. J. (1980) 192, 959--962] have cast doubt on the physiological role of malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. The high Ki values obtained in the cited studies are shown to be due to incubation conditions that cause substrate depletion, destruction of malonyl-CoA or generation of excessively high concentrations of unbound acyl-CoA (which offsets the competitive inhibition of malonyl-CoA towards carnitine acyltransferase I). The present results are entirely consistent with the postulated role of malonyl-CoA as the primary regulatory of fatty acid synthesis and oxidation in rat liver.     

Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).     

Validity of the jack-knife technique for analysing enzyme kinetic data.

An observation by Duggleby [Biochem. J. (1979) 181, 255-256] that estimates of kinetic parameters by the jack-knife technique [Cornish-Bowden & Wong (1978) Biochem. J. 175, 969--976] are sometimes outside the range of estimates from which they are calculated has been examined. No significant correlation has been found between the occurrence of this behaviour and the actual quality of the estimates.     

Activation of the Sendai virus fusion protein (f) involves a conformational change with exposure of a new hydrophobic region

The F protein of paramyxoviruses is actively involved in the induction of membrane fusion. This fusion may be between viral and cellular membranes, as in the initiation of infection or in virus-induced lysis of erythrocytes, or between the plasma membranes of different cells. The F protein is activated by proteolytic cleavage to yield two disulfide-linked polypeptides (F1 and F2); however, its mechanism of action is not clear. In the present study, the conformations of the inactive, uncleaved precursor of glycoprotein (F0), and the active, cleaved form (F1,2) have been compared. The UV circular dichroism spectra of the two forms of the F protein indicate that cleavage results in a conformational change. Detergent-binding studies by velocity sedimentation analysis of Triton X-100-protein complexes revealed an increase in exposed hydrophobic surface of the protein on cleavage. The inactive F0 bound an estimated 27 molecules of Triton X-100/F polypeptide; these molecules are presumably bound to the hydrophobic region of the glycoprotein that anchors the spike-like protein in the virus membrane and that is common to both forms of F. The active form, F1,2, bound 67 molecules of Triton X-100. This increase in the number of detergent binding sites upon F protein activation indicates the presence of a hydrophobic region that is peculiar to the active form, and that may be of functional significance in the membrane fusion reaction.     

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein)--haptoEGFPs--which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.     

Sendai virus F glycoprotein induces IL-6 production in dendritic cells in a fusion-independent manner

We previously reported that inactivated Sendai virus particle (hemagglutinating virus of Japan envelope; HVJ-E) has anti-tumor effects by eliciting IL-6 production in dendritic cells (DCs). In the present study, we investigated which components of HVJ-E elicit IL-6 production. HVJ-E containing F0 protein inactive for virus envelope-cell membrane fusion enhanced IL-6 production. Reconstituted liposomes containing F protein stimulated IL-6 production. The antibody against F protein inhibited IL-6 secretion by HVJ-E. When carbohydrate chains of the F glycoprotein were removed, HVJ-E lost the ability to stimulate IL-6 secretion. These results suggest that F glycoprotein is required for IL-6 production in DCs.     

Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell-cell fusion and a decreased dependency on the HN attachment protein for activity

Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.     

A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A is unable to process the precursor fusion glycoprotein of Newcastle disease virus.

RPE.40, a mutant strain of CHO-K1 cells isolated for resistance to Pseudomonas exotoxin A and cross-resistant to alphaviruses, is also highly resistant to virulent strains of Newcastle disease virus. The resistance of RPE.40 cells to Newcastle disease virus results from the failure to cleave the viral envelope precursor glycoprotein Fo to fusion glycoprotein F1 at the consensus sequence (Lys/Arg)-Arg-Gln-(Lys/Arg)-Arg.