Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus

The glycoproteins of Sendai virus have been isolated by a procedure involving extraction with the nonionic detergent Triton X-100 and affinity chromatography on fetuin-Sepharose. The largest Sendai virus glycoprotein (MW ~69,000) possesses both hemagglutinating and neuraminidase activities, and has been designated HN.Virions grown in MDBK cells or in the allantoic sac of the chick embryo contain similar amounts of the HN glycoprotein, but differ in their content of the other glycoproteins. Virions grown in MDBK cells contain a large amount of a glycoprotein designated F₀ (MW ~65,000). This glycoprotein is a precursor of a smaller virion glycoprotein, F (MW ~53,000) which is present in only small amount in MDBK cellgrown virions. F₀ can be cleaved to yield F by treatment of virions with trypsin in vitro. Sendai virions grown in the chick embryo lack the precursor protein F₀, but contain a large amount of F; in this case, proteolytic cleavage of F₀ to yield F occurs in ovo.The Sendai virions grown in MDBK cells, which are deficient in the small glycoprotein F, lack hemolyzing and cell-fusing activities and cannot infect MDBK cells. Virions grown in the chick embryo, which contain much F, possess both these activities and can infect MDBK cells as well as the chick embryo. MDBK cell-grown virions acquire hemolyzing and cell-fusing activities and become infective for MDBK cells when the precursor glycoprotein F₀ is cleaved in vitro to yield F.The results indicate that the small glycoprotein of paramyxoviruses is biologically active and is involved in virus-induced hemolysis, cell fusion, and the initiation of infection. The precise mechanism by which this glycoprotein participates in these reactions remains to be determined, but is now amenable to experimentation. The precursor of this glycoprotein is biologically inactive, but is incorporated into virions grown in some host cells; it may be activated by proteolytic cleavage either in vivo or in vitro. The present results provide a biochemical basis for previously observed host-dependent variation in infectivity, and in hemolysis and cell-fusion induced by paramyxoviruses.     

Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes

Mumps virus was adapted to growth in Vero cells, which yielded virus of high infectivity titers. The structural polypeptides of purified virions grown in Vero cells were similar to those described previously for egg-grown mumps virus: L (200K), HN (79K), NP (72K), F₁ (61K), P (45K), M (40K), and F₂ (16K). We have analyzed the synthesis of viral polypeptides in Vero cells by pulse labeling with radioactive amino acid precursors. The nucleoprotein (NP) was the first to be detected intracellularly above the cellular protein background at 6 h.p.i. By 12 h.p.i., all viral polypeptides were observed except for the glycoproteins F₁ and F₂, which are derived from a precursor designated F₀ (74K). Two low-molecular-weight polypeptides not present in purified virions were also detected in infected cells. They are designated pI (28K) and pII (19K). Peptide mapping revealed that these two polypeptides share regions of their amino acid sequence and that they are also related to the structural protein P. Polypeptides pI and pII were found in several cell types (Vero, CEF, MDBK cells) infected with mumps virus. Infection of Vero cells with other paramyxoviruses (SV5 and Sendai virus) did not induce the synthesis of proteins comparable to pI and pII, whereas in mumps virus-infected cells no counterpart to the nonstructural C protein of Sendai virus was detected. Pulse-chase experiments suggest that pI and pII may not be derived from P by proteolytic cleavage.     

Determination by peptide mapping of the unique polypeptides in Sendai virions and infected cells.

Peptide mapping of all of the Sendai virion polypeptides and the viral polypeptides synthesized in infected cells has shown that C (MW ～22,000), which has been found only in infected cells, is unique and is thus a candidate for a nonstructural polypeptide. It has also been found that the L polypeptide of Sendai virus is unique, that polypeptide 5 is derived from NP, that polypeptide A is host-cell actin, and that the polypeptides L, P, HN, F₀, NP, and M have the same peptide composition in virions as in infected cells.     

Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide HA

The cleavage of the HA polypeptide, the largest glycoprotein of influenza virus, to polypeptides HA1 and HA2 has been studied using the WSN strain of influenza A₀ and the RI/5^? strain of influenza A₂ grown in different host cells. Cleavage of the HA polypeptide is not required for the assembly of infectious, hemagglutinating virions. Cleavage is both strain dependent and host cell dependent, and correlates with the extent of cell damage, suggesting that the enzymes involved are host cell specified. Virions grown in MDBK cells without calf serum in the medium contain almost entirely uncleaved HA polypeptides. Virions harvested early in the growth cycle contain more uncleaved HA polypeptide than virions harvested late from the same cells when cytopathic effects are extensive. The proteolytic nature of the cleavage has been demonstrated in vitro with trypsin. Comparison of the specific hemagglutinating activity and infectivity of virions which contain different amounts of the uncleaved HA polypeptide and the cleavage products HA1 and HA2, and of the capacity of such virions to react with the soluble glycoprotein receptor substance fetuin, have shown that uncleaved HA polypeptides are as active in hemagglutination and adsorption to cellular and soluble receptors as are the disulfide-bonded complexes composed of HA1 plus HA2. The proteolytic cleavage of the HA polypeptide to HA1 and HA2 is thus not required for virus assembly or for full expression of the biological properties of the virion. Rather, it appears to be a nonessential result of events occurring in infected cells which are undergoing cytopathic effects.     

The site of cleavage in infected cells and polypeptides of representative paramyxoviruses grown in cultured cells of the chorioallantoic membrane

Summary Cultured cells of the chorioallantoic membrane (CAM) fulfilled the need of using the same cell system that was permissive for representative paramyxoviruses to carry out studies on the biosynthesis of their glycoproteins in infected cells.The polypeptide composition of the respective paramyxoviruses [Newcastle disease virus (NDV), paramyxovirus Yucapipa (PMY), and Sendai virus], grown in eggs and CAM-cells, was essentially identical. In egg-grown PMY a large glycoprotein (LGP) was present but only in some CAM-grown preparations of virus labeled with [³H]-glucosamine and rarely in [³⁵S]-methionine or [³H]-amino acids (valine, leucine, and tyrosine) labeled viruses.The site of cleavage of precursor F₀ to F_1,2 was not the same. In contrast to the cleavage of Sendai virus glycoprotein, cleavage was intracellular in NDV and PMY infected cells. Homologous antisera against the glycoproteins failed to inhibit cleavage of HN₀ or F₀ in cells infected with the representative paramyxoviruses.With 8 FiguresOn leave from: Department of Microbiology, California State University—Los Angeles, Los Angeles, CA 90032, U.S.A.     

Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide.

The infectivity of virions of the WSN strain of influenza A ($\text{A}\text{WSN}$) and the 1760 strain of influenza B (B/1760) which possess their hemagglutinin in the form of the uncleaved HA polypeptide can be enhanced as much as 100-fold by proteolytic cleavage of HA to yield HA₁ and HA₂. Hemagglutinating activity is unaffected by cleavage. The HA polypeptide of $\text{A}\text{WSN}$ virions is susceptible to cleavage by trypsin and plasmin, whereas that of B/1760 virions is resistant to plasmin. The increase in infectivity can be demonstrated in different cell types, i.e., MDBK, BHK21-F, CEF, or HKCC cells, as well as in the chick embryo, and in titrations in both liquid medium and plaque assays under agar. Chymotrypsin cleaves the HA polypeptide of WSN virions in the same general region of the molecule as trypsin and plasmin, but without the enhancement of infectivity produced by the latter enzymes. Incubation of WSN virions with chymotrypsin prevents the enhancement by subsequent treatment with trypsin, but the increase in infectivity caused by an initial treatment with trypsin is not abolished by subsequent treatment with chymotrypsin. These results indicate that enhancement of infectivity of influenza virions results from proteolytic cleavage at a specific site on the HA molecule; however an increase in infectivity is not associated with cleavage at a nearby but different site.Both $\text{A}\text{WSN}$ and B/1760 virions are capable of undergoing multiple cycles of replication in cells under conditions in which no cleaved HA polypeptides are detected on the virions produced. These results are compatible with the enhancement of infectivity being the result of an increase upon cleavage in the efficiency of some stage in the initiation of infection beyond the initial adsorption step. The increase in infectivity caused by proteolytic cleavage of the HA polypeptide provides a biochemical explanation for the previously observed enhancement of plaquing efficiency of influenza viruses by the inclusion of pancreation or trypsin in the agar overlay.     

The polypeptides of canine distemper virus: Synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses

The biochemical and immunological properties of the polypeptides of canine distemper virus (CDV), their synthesis and processing in infected cells, and their relatedness to the polypeptides of other morbilliviruses have been studied. CDV virions contain six major polypeptides which are analogous to those of measles virus (MV). These polypeptides with their estimated molecular weights (m_r) are: L (200,000); H (76,000); P (66,000); NP (58,000); F (62,000), which consists of two disulfide-linked polypeptides, F₁ (40,000) and F₂ (23,000); and M (34,000). The H, F₁, and F₂ polypeptides of CDV are glycosylated; the presence of carbohydrate on F₁ is in contrast to its absence on the F₁ of MV. The CDV F₂ has a larger apparent M_r than the MV F₂ (23,000 vs 12,000). The NP and P polypeptides of CDV are phosphorylated, and in pulse-chase experiments in CDV-labe;ed cells the P polypeptide was rapidly lost. In addition to the structural polypeptides, a putative nonstructural protein, NS (M_r 18,000), was found in CDV-infected cells. The polypeptide also turned over rapidly in pulse-chase experiments.The immunological relatedness of the polypeptides of MV and CDV and of two other morbilliviruses, rinderpest (RV) and a bovine encephalitis virus (107) was shown by immuno-precipitation of the viral polypeptides from CDV- and MV-infected cells with antisera against each of the four viruses. The only failure to exhibit reciprocal reactivity between CDV and MV was found with the H polypeptides, where only a one-way cross was found, i.e., MV antiserum precipitated all of the CDV polypeptides, whereas CDV antiserum precipitated all of the MV polypeptides except H. RV antiserum resembled that of MV; it precipitated all of the polypeptides of both MV and CDV, whereas 107 antiserum, like that of CDV, precipitated all of the CDV polypeptides and all of the MV polypeptides except H. These results indicate that these four morbilliviruses with different host ranges are antigenically closely related, with MV apparently more closely related to RV, and CDV to 107 virus. In spite of their antigenic similarities, the individual polypeptides of CDV and MV could be easily distinguished by peptide mapping. Some similarities were found in the internal polypeptides P, NP, and M, but very little in the surface glycoproteins, H and F₁.     

The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides.

Phosphorylated and unphosphorylated forms of the membrane polypeptide (M) and the nucleocapsid polypeptide (NP) of Sendai virus have been identified in both infected cells and virions. Polypeptide B, found previously in infected cells, has been shown to be a phosphorylated form of M by peptide mapping, by conversion of M to B by phosphorylation in both pulse-chase experiments in infected cells and in vitro by a virion-associated protein kinase and, conversely, by conversion of B to M through the loss of phosphate in cell lysates. Although very little of the phosphorylated form was found in virions, B and M were found in similar proportions in infected cells after a 30-min pulse, suggesting that the nonphosphorylated form is preferentially incorporated into virions or that phosphate is removed during the maturation process. The finding of phosphorylation of M and NP in cells suggests that this may play a role in virus replication or assembly. Two phosphorylated forms of the nucleocapsid polypeptide (NP_P1 and NP_P2) have been found, but the unphosphorylated NP is the predominant form in both cells and virions. The similarity of these three polypeptides, except for their phosphate content, has been shown by peptide mapping. The membrane polypeptides B and M were separated by electrophoresis on polyacrylamide gels in the absence of urea, but in the presence of 4 M urea they comigrated. In contrast to these results with polypeptides B and M, polypeptides NP, NP_P1 and NP_P2 were resolved in gels in the presence of 4 M urea but comigrated in its absence. Thus, the behavior of viral phosphopolypeptides in different polyacrylamide-gel systems varies depending on the polypeptides. Possible biological roles of phosphorylation of the virus polypeptides are discussed.     

Structural polypeptides of mumps virus.

The structural polypeptides of egg grown mumps virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. Mumps virions contained eight major polypeptides with mol. wt. of 75, 73, 71, 61, 47, 44, 42 and 40 X 10(3). The 75 K and 61 K polypeptides were glycosylated. In virions treated with pronase and trypsin, the 75 K glycoprotein was removed more readily from the virus than the 61 K glycoprotein. The gradual removal of the 75 K glycoprotein was paralleled by a decrease of haemagglutinating activity. The large glycoprotein was cleaved into a 40 K glycoprotein by trypsin treatment. Pronase and trypsin treatment also removed the smallest 40 K non-glycosylated polypeptide. Thus this polypeptide appears to be located on the outside of the virion and probably represents a cleavage product of the large glycoprotein. Treatment of virions with 2% Triton-X 100 under alkaline conditions in the absence or presence of 2 M-KCl solubilized the two glycoproteins and a fraction of the 71 and 44 K polypeptides, but not the 73 and 47 K polypeptides. The two smallest polypeptides were solubilized by treatment with 2% Triton X-100 in the presence of 2 M-KCl. Since the 40 K polypeptide was interpreted to represent a cleavage product of the large surface glycoprotein the 42 K polypeptide was proposed to represent the membrane protein of mumps virus. The 44 K polypeptide co-migrated with Vero cell actin. The nature of the 47 K polypeptide could not be determined, but it is probably located in the central part of the virus. The 73 K polypeptide and in some experiments also the 71 K polypeptide were found in purified nucleocapsid preparations. It is concluded that mumps virus has a general polypeptide composition similar to other paramyxoviruses. However, the molecular weights of the different polypeptides of mumps virus differ markedly from the corresponding polypeptides in Newcastle disease virus and Sendai virus.     

Protease activation mutants of sendai virus. Activation of biological properties by specific proteases.

A new class of Sendai virus mutants (pa mutants) is described that exhibit altered specificities with respect to protease activation of infectivity and altered host range. Sendai virus requires proteolytic cleavage of a virion glycoprotein (F₀ to F) in order to be infective. Wild-type virus can be activated in vitro by treatment with trypsin, but not chymotrypsin or elastase, or in vivo by addition of trypsin to cells, e.g., MDBK, which lack activating protease. Mutants have been isolated that are activated by chymotrypsin (pa-c mutants) or elastase (pa-e mutants). Some mutants are no longer activated by trypsin, and these mutants have lost the ability to undergo multiple-cycle replication in the embryonated chick egg unless chymotrypsin or elastase is added to the mutants also activate hemolysis. These findings with pa mutants support the previous conclusion based on results with wild type virus, that a host-dependent cleavage of the F₀ protein is required for the infectivity of Sendai virus and for activation of hemolyzing and cell-fusing activities. The results obtained indicate that the host range and tissue tropism of Sendai virus are determined at least in part by the availability of the appropriate protease required for activation of infectivity.     

Measles virus polypeptide synthesis in infected cells

The synthesis of measles virus polypeptides has been studied using an inhibitor of virus-induced cell fusion, carbobenzoxy-d-phenylalanyl-l-phenylalanyl-nitro-l-arginine (SV4814). Cells infected at a multiplicity of 10 fuse extensively by 17 hr and die shortly thereafter, making it difficult to detect viral polypeptides. Cells protected from fusion by SV4814 survive and continue to produce virus, and the synthesis of viral polypeptides can be followed for 4 days. The previously described measles virion polypeptides G, 2, NP, 5, and M have been identified in infected cells, and, in addition, a polypeptide (L) with a molecular weight of ～200,000 has been found in infected cells and in small amounts in virions. (New designations suggested for polypeptides G, 2, and 5 are H, P, and F₁, respectively.) A glycosylated polypeptide (F₀, MW ～62,000) has also been found in infected cells, but not in virions. This polypeptide is thought to be the precursor of two polypeptides which appear under pulse-chase conditions: F₁ (MW ～40,000), which is not glycosylated, and F₂, a small glycosylated polypeptide detected with [³H]glucosamine labeling. In addition to facilitating studies of measles virus polypeptide synthesis, the use of SV4814 has shown that cell fusion is the major factor in early cell death caused by measles virus, but that cell death ultimately ensues in the absence of cell fusion, indicating another mechanism of measles virus-induced cell damage.     

Charge heterogeneity in polypeptides of negative strand RNA viruses

We surveyed charge heterogeneity in the polypeptides of three enveloped RNA viruses, Sendai virus, influenza virus (WSN strain), and vesicular stomatitis virus (VSV). Isoelectric focusing in polyacrylamide gel was followed by electrophoresis in a second dimension after denaturation of the polypeptides with sodium dodecyl sulfate. Nucleocapsid polypeptides P and NP of Sendai virus exhibited charge heterogeneity which may correspond to various extents of post-translational phosphorylation (R. A. Lamb and P. W. Choppin, 1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology81, 382–397. Nucleocapsid polypeptide N of VSV was more homogeneous, whereas the NP polypeptide of influenza virus appeared to be too basic (isoelectric point greater than 8.0) to be resolved in the isoelectric focusing system employed. Glycosylated envelope polypeptides of all three viruses separated into multiple (3 to 8) acidic species with isoelectric points in the range of 4 to 5 for VSV glycopolypeptide G and 5 to 6 for the glycopolypeptides of Sendai virus and influenza virus. Although some of the heterogeneity in VSV glycopolypeptide G may stem from variations in content of N-acetyl neuraminic acid (NANA), a different basis for the heterogeneity is suggested by failure of extensive neuraminidase treatment to abolish it. Moreover, NANA is removed from the glycopolypeptides of Sendai virus and influenza virus by virus-specified neuraminidases. A possible relationship between content of amino sugars (other than NANA) and charge heterogeneity was suggested by the finding that the amount of radioactive glucosamine incorporated biosynthetically into G of VSV and HN of Sendai virus was greater in the more electropositive species. The functional consequences of this variety of post-translational modifications remain to be determined.     

Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site

Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F1 and F2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P.W. Choppin [1976). Virology, 265-277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60-70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.     

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN₀ and F₀, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN₀ and F₀ together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN₀ was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN₀ and F₀ with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN₀ can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F₀ shows a specific requirement for trypsin.     

The polypeptides of human parainfluenza type 2 virus and their synthesis in infected cells

Summary We have studied the structural components of three strains of human parainfluenza virus type 2 (HPI-2) and identified the virus-specific polypeptides. Molecular weight of P and F1 polypeptides determined by us, when compared with that reported previously, was in reversed order. HN polypeptide existed chiefly as disulfide-linked dimers in cells infected with Toshiba strain, while as monomers and dimers in nearly equal proportion in cells infected with two other strains. Similar disulfide-linked NP oligomers were found in cells infected with all three strains. F1 and F2, cleaved forms of F protein, could be detected in cells infected with all three strains, but the ratio of cleaved (F1 and F2) to uncleaved (F₀) forms was markedly lower in 62-M 786- and 63-M 1-infected than in Toshiba strain-infected cells. However, there was no difference of oligopeptide mapping patterns and isoelectric point of F polypeptide between Toshiba and 62-M 786 strains. By contrast, oligopeptide mapping patterns of HN protein of Toshiba strain differed from those of the two other strains. Furthermore, the HN polypeptide of Toshiba strain was phosphorylated in the infected Vero cells, but that of the other two strains was not.     

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P₁, P₂, P₃), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus.The three high molecular weight polypeptides, P₁, P₂, and P₃, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.     

Biogenesis of vaccinia: evidence for more than 100 polypeptides in the virion.

The polypeptides of vaccinia were separated and analyzed by two-dimensional (2-D) gel electrophoresis patterned after that of O'Farrell (1975). Following labeling with [³⁵S]-methionine, [³³P]phosphate, or [³H]glucosamine, pure virions were dissociated and subjected to electrophoresis using either isoelectric focussing or nonequilibrium pH gradient conditions in the first dimension and SDS-polyacrylamide slab gels in the second dimension. By this means at least 111 spots, of which 7 or more were basic proteins, were resolved in the autoradiograms. Authenticity of several single spots was established. This included a glycoprotein of molecular weight 34,000 (34 K) labeled with [³H]glucosamine; a phosphorylated basic protein of about 11 K marked with ³³PO₄; isolated purified surface tubular elements of 58 K; two major core polypeptides of 60 and 62 K derived from larger precursors after proteolytic cleavage; a precursor polypeptide of 25 K known from previous studies with a ts mutant 1085 to undergo cleavage; and an 18 K polypeptide which appears in wild type and ts 1085 infections under circumstances permissive for cleavage. The 2-D analysis therefore reveals that poxviruses are in terms of their polypeptides, even more complex than had been anticipated previously.     

Isolation and characterization of the measles virus F1 polypeptide: comparison with other paramyxovirus fusion proteins

Measles virus fusion (F) protein has been isolated by immunoadsorption to a complex of monoclonal antibodies bound to protein A-Sepharose. The 41-kDa F1 component of the fusion protein was obtained pure in high yield by preparative SDS-polyacrylamide gel electrophoresis. The amino acid composition of the F1 chain was determined and the N-terminal sequence was analyzed for 40 residues. The structure determined is largely hydrophobic, with 24 residues of Val, Ile, Leu, Met, Phe, or Ala. Comparison with previously published data on the F1 polypeptide of Sendai virus showed considerable similarity in amino acid composition. Extensive N-terminal sequence homologies with F1 polypeptides of different paramyxoviruses are also noticed, including a nine-residue segment strictly conserved among four F1 polypeptides studied, as well as a weaker but distinct and Gly-rich sequence homology with the influenza A and B virus HA2 polypeptides. The evolutionary conservation of the N-terminal region at the site of cleavage of surface glycoproteins of the two families of myxoviruses highlights its specialized function in membrane fusion.     

Japanese encephalitis virus glycoproteins

Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK₂ cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.     

Host induced modifications of Newcastle Disease Virus virion polypeptides

Summary The polypeptide composition of Newcastle Disease Virus (NDV) virions grown in two host cell cystems—chorioallantoic membrane (CAM) and BHK-21 cells—was studied. Two strains of virus were compared, one highly virulent, the other completely avirulent. No significant differences in the polypeptide composition of the two strains of virus could be detected. However, differences were found in virions grown in different hosts, the same differences being found in both strains. An additional polypeptide is found in BHK grown virus which is not present in CAM grown virus and this is associated with a decreased relative amount of nucleocapsid protein in BHK grown virus. The possibility of this new polypeptide being a degradation product of the nucleocapsid protein is discussed. BHK grown virions also contain increased amounts of a polypeptide migrating to a position which might be expected of the F₀ precursor glycoprotein. However, in contrast to the F₀ polypeptide, this polypeptide does not appear to be glycosylated.With 4 Figures