Studies on the development of the charge heterogeneity of the influenza virus glycoproteins

Summary The heterogeneity in charge of the influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA) is retained, when glycosylation is inhibited by tunicamycin (TM) or 2-deoxyglucose (2-dg). This is in contrast to the charge heterogeneity of the G protein of vesicular stomatitis virus (VSV), which is mainly due to heterogeneous sulfation of the carbohydrate side chains and therefore is abolished by the above mentioned inhibitors of glycosylation. Thus, the charge heterogeneity of influenza virus glycoproteins might be attributable to some as yet unidentified modifications of the polypeptide backbone.With 3 Figures     

Synthesis of Sendai virus polypeptides by a cell-free extract from wheat germ.

The '18S' RNA population from Sendai virus-infected cells efficiently directed the synthesis of three virus-specific polypeptides P, NP and M, in wheat germ cell-free extracts. In agreement with previous results obtained with a reticulocyte extract, there was little or no production of virus glycopolypeptides. Analyses of tryptic peptides revealed close correspondence between the primary structures of NP and M made in vitro and the authentic virus polypeptides.     

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.     

Respiratory syncytial virus proteins

Respiratory syncytial (RS) virus grown in BS-C-1 cells was concentrated from the fluid of infected cultures by precipitation with polyethylene glycol (PEG) and banded by isopycnic centrifugation in sucrose or metrizamide density gradients. At least six virus-specified polypeptide bands, one of which was heterogeneous, could be resolved by continuous SDS-polyacrylamide gel electrophoresis (PAGE) and an additional band by discontinuous SDS-PAGE. The three predominant viral polypeptides were a glycopolypeptide of 48 × 10³ (VGP48), a nucleocapsid polypeptide of 41 × 10³ (VP41), and a polypeptide of 27 × 10³ molecular weight (VP27). Three minor viral polypeptides have been assigned the molecular weight of 38 × 10³ (VP38), 32 × 10³ (VP32) and 25 × 10³ (VP25). A minor glycopolypeptide of molecular weight 42 × 10³ (VGP42) may exist also. Partial purification was accompanied by the loss of high molecular weight glycopolypeptides; however, one high molecular polypeptide (P2) remained consistently associated with the presumptive polypeptides and may represent an eighth virus-specified polypeptide.VP27 can be obtained in relatively pure form by sedimentation of detergent-treated RS virus in a metrizamide gradient containing detergent.     

Structural polypeptides of the murine coronavirus DVIM

Summary The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.With 3 Figures     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. I. Gel filtration in 6 M guanidine hydrochloride.

Highly purified glycopolypeptides HA1 and HA2 were separated from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2) by gel filtration in 6 M guanidine hydrochloride under reducing conditions. The purity of both glycopolypeptides was proved by extensive studies. Despite the lack of C-terminal end, the isolated HA2 glycopolypeptide displayed some hydrophobic properties.     

Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses.

The paramyxovirus glycoprotein which is required for virus-induced cell fusion, hemolysis, and the initiation of infection (F protein) has been found with three different viruses to consist of two disulfide-linked glycopolypeptide chains (F₁ and F₂) which are derived from the precursor glycopolypeptide (F₀) by proteolytic cleavage. The larger glycopolypeptide chain (F₁) previously identified in SV5, Sendai, and Newcastle disease virions has an estimated molecular weight of 48,000–54,000. The smaller polypeptide chain (F₂) has been found to have a molecular weight of ～10,000–16,000, depending on the virus. The identification of the F₂ polypeptide was accomplished by isolating the disulfide-linked complex (F_1,2) under nonreducing conditions, followed by reduction of the disulfide bonds and separation of the two polypeptide chains. Although both polypeptides are glycosylated, the F₂ polypeptide contains more carbohydrate per unit protein than F₁. No free N-terminus could be detected on the F₀ or F₂ polypeptides of Sendai virus, whereas N-terminal phenylalanine was found on F₁. This suggests that the order of the F₀ polypeptide is X-NH-F₂—Phe-F₁—COOH. The finding that with three different paramyxoviruses the biologically active virions possess F₁ and F₂ polypeptides suggests that this is a general feature of paramyxoviruses, and that the activation of infectivity, cell fusion, and hemolysis is due to a conformational change in the F protein resulting from proteolytic cleavage to form an active complex of two disulfide-linked polypeptide chains.     

In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses

The messenger RNAs of SV5 and two strains of Sendai virus were isolated from infected cells and translated in a wheat germ cell-free system. Comparison of the peptide maps of the polypeptides synthesizedin vivo andin vitro established that the nonglycosylated polypeptides P, NP, and M of both SV5 and Sendai virus had been synthesizedin vitro. In immunoprecipitation studies of the putative SV5 polypeptides synthesizedin vitro, antiserum against whole virions precipitated NP, P, and M, and other polypeptides which did not comigrate with mature virion polypeptides. Monospecific antisera against the HN and F glycoproteins precipitated two of the latter polypeptides with molecular weights of ～55,000 and 50,000, respectively, suggesting that the nonglycosylated forms of these polypeptides had been synthesizedin vitro. Polypeptide C, previously found in Sendai virus-infected cells and proposed to be a virus-specific nonstructural polypeptide, has been found to exhibit strain-specific differences in migration in polyacrylamide gels. Polypeptides with the appropriate strain-specific migration have been synthesizedin vitro with mRNAs from cells infected with the different Sendai virus strains, and shown by peptide mapping to be the same polypeptides as those synthesizedin vivo. The results have thus provided further evidence that C is a virus-coded nonstructural protein. Another polypeptide (C′), with migrates slightly slower than C, has been found in infected cells and synthesizedin vitro. This polypeptide also exhibits strain-specific differences in migration in polyacrylamide gel electrophoresis, and has been shown by peptide mapping to be similar to C. The explanation for the difference in migration between C and C′ is as yet unknown.     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. III. Reactivity with human convalescent sera.

The immune reactivity to both haemagglutinin glycopolypeptides HA1 and HA2 [prepared from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2)], was demonstrated by both gel double immundiffusion and radioimmunoassay in human convalescent sera obtained after natural infection during influenza epidemics in 1974/75 and 1976/77. In gel double immunodiffusion, the precipitin line(s) corresponding to glycopolypeptide HA1 were always more distinct than precipin line(s) corresponding to glycopolypeptide HA2. In radioimmunoassay, human convalescent sera revealed higher titres for binding of 125-I-labelled HA2 than for 125-I-labelled HA1. Characterization of human convalescent sera was completed by haemagglutination-inhibition test.     

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. II. Reactivity with rabbit sera against intact virus and purified undissociated haemagglutinin.

Rabbit sera produced against either intact virus or purified undissociated haemagglutinin were examined for reactivity with highly purified haemagglutinin glycopolypeptides. Sensitive radioimmunoassay for 125I-labelled glycopolypeptides revealed antibody reactive with either glycopolypeptide HA1, or glycopolypeptide HA2. Antibodies against the carbohydrate moiety were responsible only for a part of the binding activity. Under the conditions employed, the binding activity for glycopolypeptide HA2 was much stronger than for glycopolypeptide HA1. Competition assays suggested that immune reactivities were due to distinct antibody populations (i.e. with a specifity for glycopolypeptide HA1 and glycopolypeptide HA2, respectively). The immune reactivity to both haemagglutinin constituents, glycopolypeptides HA1 and HA2, was also shown by gel double diffusion. The precipitin line(s) corresponding to glycopolypeptide HA1 was (were) usually more distinct than precipitin line(s) corresponding to glycopolypeptide HA2. The glycopolypeptides HA1 and HA2 showed the reaction of nonidentity in immunodiffusion analysis.     

Differences in the electrophoretic migration rates of polypeptides and RNAs of recent isolates of influenza B viruses

Summary The electrophoretic migration rates of structural and non-structural poly-peptides of 38 influenza B viruses isolated in epidemics in 1978–1980 and antigenically closely related to B/Singapore/222/79 virus were compared using high resolution SDS polyacrylamide gels. Thirty of the viruses could be distinguished from the prototype B/Singapore/222/79 virus by electrophoretic migration rate differences in HA, 17 by differences in NP and 27 by differences in mobility of the NS1 polypeptide. Mobility differences of NP, NS1 and HA polypeptides was noted in influenza B viruses isolated in the UK in the same year. In addition, electrophoretic mobility of³²P labelled virus RNAs varied for certain UK isolates and indicated heterogeneity in genes 2, 3, 4 and 8 coding for polymerase proteins 2 and 1, nucleoprotein (NP) and non-structural protein (NS1) respectively.With 3 Figures     

Impaired antiviral response in human hepatoma cells.

Hepatitis B, C, and D viruses can infect liver cells and in some individuals establish a chronic phase of infection. Presently, relatively little information is available on the antiviral mechanisms in liver cells. Because no good in vitro model infection systems for hepatitis viruses are available, we have used influenza A, Sendai, and vesicular stomatitis (VSV) viruses to characterize interferon (IFN) responses and IFN-induced antiviral mechanisms in human hepatoma cell lines. HepG2 or HuH7 cells did not show any detectable IFN-alpha/beta production in response to influenza A or Sendai virus infections. Treatment of cells with IFN-alpha resulted in upregulation of IFN-alpha-inducible Mx, 2',5'-oligoadenylate synthetase (OAS) and HLA class I gene expression but only with exceptionally high levels of IFN-alpha (>/=100 IU/ml). Accordingly, high pretreatment levels of IFN-alpha, 1000 IU/ml for influenza A and VSV and 100 IU/ml for Sendai virus, were required before any detectable antiviral activity against these viruses was seen. IFN-gamma had some antiviral effect against influenza A virus but appeared to be ineffective against VSV and Sendai virus. IFN-gamma upregulated HLA class I protein expression, whereas Mx or OAS expression levels were not increased. There was a modest upregulation of HLA class I expression during Sendai virus infection, whereas influenza A virus infection resulted, after an initial weak upregulation, in a clear decrease in HLA class I expression at late times of infection. The results suggest that hepatoma cells may have intrinsically poor ability to produce and respond to type I IFNs, which may contribute to their inability to efficiently resist viral infections.     

Characterization of TAP-Independent and Brefeldin A-Resistant Presentation of Sendai Virus Antigen to CD8+ Cytotoxic T Lymphocytes

H-2K^b-transfected T2 cells, which lack both TAP1/2 and LMP2/7 genes, are able to efficiently process and present Sendai virus Antigen to K^b-restricted Sendai virus-specific CTL. This presentation is not inhibited by Brefeldin A (BFA). Here we extend our analysis of this novel antigen presentation pathway. We show that presentation of Sendai virus antigen was not due to sensitization of T2K^b cells by peptides in the virus preparation or peptides released from virus infected cells. Also, the ability to present Sendai virus in a BFA resistant fashion was specific for cells of the T2 lineage. Re-expression of TAP1/2 genes in T2K^b cells did not alter the capability to present antigen in a BFA resistant fashion, i. e. the presence of a functional TAP transporter complex did not relocate (all) peptides to the classical pathway for antigen processing and presentation. We found that co-infection of T2K^b cells with either Sendai virus plus influenza virus or Sendai virus plus VSV did not relocate presentation of influenza or VSV antigen to the TAP independent BFA resistant antigen presentation pathway. Peptide elution experiments and studies with peptide-specific CTL firmly demonstrated that the antigen presented by T2K^b cells after infection with Sendai virus was the natural Sendai virus epitope NP324-332. The same epitope, when expressed as a minigene in vaccinia virus, could be presented also by T2K^b cells but this presentation could be blocked by BFA. Thus, the TAP independent BFA resistant presentation of antigen seem cell (T2 lineage) and virus (Sendai virus) specific, but not epitope specific. The ability of T2K^b cells to present Sendai virus antigen in a TAP independent BFA resistant fashion was only partially blocked by lysosomal inhibitors such as methylamine, ammonium chloride and chloroquine. These findings demonstrate that TAP1/2-independent and BFA-resistant class I processing is only expressed in certain cell types, in parallel with classical MHC class I processing, and that Sendai virus selectively can enter this pathway. Hypothetical models for the TAP-independent class I processing are discussed.     

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.     

Isolation of subviral components from transmissible gastroenteritis virus.

Exposure of purified transmissible gastroenteritis virus, a porcine coronavirus, to non-ionic detergents resulted in the removal of the surface projections and greater than 98% of the virus lipid. Virus RNA was associated with a subviral particle which had a sedimentation coefficient of 650S, compared with 495S for the intact virion, and which banded in Cs2SO4 gradients at 1-295 g/ml. Negatively stained preparations of subviral particles were shown by electron microscopy to contain spherical particles of 60 to 70 nm diam., similar in appearance to those derived from oncornaviruses. Polyacrylamide gel electrophoresis of the polypeptides from isolated subviral particles showed that these structures contained three of the four major virus structural proteins, the arginine-rich polypeptide VP2 and the two membrane glycopolypeptides VP2 and 4. The detergent-liberated surface projections, composed of a single species of sulphated glycopolypeptide, VPI, were isolated by rate-zonal centrifugation through sucrose gradients followed by precipitation with ammonium sulphate in the presence of bovine serum albumin.     

Intracellular metabolism of sendai virus nucleocapside.

We examined the intracellular pathways that Sendai virus polypeptides P and NP take into nucleocapsids by use of pulse-chase labeling and cell fractionation. Although most molecules of the major polypeptide NP entered nucleocapsids by way of a large soluble pool with slow exit kinetics, a fraction of NP molecules appeared to bypass this pool, entering nucleocapsids directly after synthesis. In contrast, all molecules of the minor polypeptide P passed directly into nucleocapsids, never appearing in a soluble pool. These data indicate that Sendai virus nucleocapsid assembly occurs in two steps: condensation of NP with viral genomic RNA followed by addition of P (and probably L) to the structure. The data also explain, in part, why newly synthesized P is rapidly incorporated into virions (Portner, A., and Kingsbury, D. W. (1976). Virology73, 79–88). We identified a population of nucleocapsids that had previously been unrecognized. This fraction, representing about half of all intracellular nucleocapsids, sedimented at low centrifugal forces, as if attached to a large cellular organelle such as the cytoplasmic cytoskeleton. The labeling data suggested that these “bound” nucleocapsids, rather than the “free” nucleocapsids, were the source of supply for virion assembly.     

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.     

Rescue of a Sendai virus DI genome by other parainfluenza viruses: implications for genome replication.

Using a defective interfering Sendai virus stock (DIH4) freed of nondefective helper virus, we found that the closely related parainfluenza viruses 1 and 3 could substitute for the Sendai virus helper in replicating DIH4, creating chimeric nucleocapsids. The morbillivirus measles and the rhabdovirus VSV could not substitute. When DIH4 is incubated intracellularly for 5 days in the absence of help, the ability of PIV3 to rescue DIH4 at this time depended on fresh Sendai virus polymerase. The PIV3 polymerase apparently can only copy the chimeric template, but not that wrapped in the homologous Sendai NP protein. These results suggest that the cis-acting RNA sequences important for genome replication, e.g., the promoter and the encapsidation site, have been conserved among these viruses, but that the interactions between the polymerase and the template protein NP are unique for each virus.