The purification of rubella virus (RV) and determination of its polypeptide composition

Rubella virus (RV) has been propagated in murine fibroblasts (L cells) and purified using two Renografin gradients. The virus was grown in the presence of 2 μCi/ml [³H]uridine, pelleted from tissue culture media 6 days postinfection, and applied to a 25–45% discontinuous gradient. A single, sharp band was observed at the interface. This band was collected and applied to a 30–45% continuous gradient which separated intact labeled virions from ³H-labeled, light density material. Infectivity was measured using a modified hemadsorption assay. A recovery of 90% of the RV infectivity was achieved by these methods. Purified virions obtained in this way were dissociated, labeled with ¹²⁵I, immune precipitated with rubella-specific antiserum, and subjected to polyacrylamide slab gel electrophoresis and autoradiography. Four polypeptides were observed with molecular weights of 44, 41, 24, and 19 × 10³ designated as VP44, VP41, VP24, and VP19, respectively.     

The effect of dibutyryl cyclic AMP on restricted replication of rubella virus in rat glial cells in culture

Normal adult rat glial (RG) cells in culture have been shown to restrict rubella virus (RV) replication, permitting the synthesis of only five of the seven intracellular viral polypeptides associated with a normal, productive infection. Recently uninfected RG cells have been reported to differentiate into mature glia in the presence of dibutyryl cyclic AMP (dB-cAMP), suggesting that the RG cells may be a continuous cell line of undifferentiated glioblasts. This communication describes the effect of dB-CAMP on rubella-infected RG cells. Following exposure to dB-CAMP, RV antigens could be demonstrated in the cytoplasm of the differentiating glioblasts, all seven intracellular RV polypeptides could be detected following immune precipitation, slab gel electrophoresis, and autoradiography, and infectious progeny virions could be titered from the tissue culture medium. These data indicate that dB-cAMP promotes glioblast maturation and a concomitant activation of RV replication.     

Antigenic differences among multiply charged Moloney murine leukemia virus p30 polypeptides found inside infected cells

At least three Moloney murine leukemia virus (M-MuLV) p30 polypeptides (p30's), viz., a major species at pI 6.3 and two minor ones at pI 6.1 and pI 6.6, have previously been identified in purified virions by 2-dimensional gel electrophoresis and chromatofocusing (Katoh, I., Yoshinaka, Y. and Luftig, R.B. (1984) J. Gen. Virol. 65, 733-741). We have observed a similar, but distinctive pI pattern for [35S]methionine-labeled MuLV p30's in lysates from chronically infected (MuLV) cells. The variation in pI pattern of the intracellular MuLV p30's was dependent on the type of p30 reactive antibody used for immunoprecipitation. Specifically: a p30 spot with pI 6.3 was always precipitated as the major spot with three different antibodies, minor spots with pI 6.0 and 6.6 were variably seen dependent on the antibody used, and an intracellular p30 spot at pI 6.1 was only precipitated with a rat p30 monoclonal antibody but not with monospecific mouse or intact MuLV cross-reacting p30 sera. These results indicate that first, there are differences between the pI pattern of virion and intracellular MuLV p30's, and second, the antigenic determinants of intracellular p30's vary dependent on the antibody used for immunoprecipitation.     

The polypeptides of human respiratory syncytial virus: products of cell-free protein synthesis and post-translational modifications.

The properties and inter-relationships of virus-induced polypeptides synthesized in BS-C-1 cells infected with human respiratory syncytial (RS) virus have been investigated in vivo and in vitro. Analysis of the kinetics of synthesis of RS virus-induced polypeptides in vivo provided no evidence for temporal controls, a situation comparable to that observed with other negative-stranded RNA viruses with unsegmented genomes. In vitro translation of cytoplasmic RNA extracted from infected cells resulted in the synthesis of six virus-induced polypeptides, four of which were of similar molecular weights to species produced in infected cells. Synthesis of all of these polypeptides was directed by polyA)-containing RNA. The identity of the major nucleocapsid polypeptide (VP 41) synthesized in vitro was confirmed by peptide mapping. A prominent polypeptide in infected cells, VP 38, was not synthesized in vitro. The relationship of this polypeptide to other RS virus polypeptides was investigated in detail, and it was observed that VP 38 and VP 41 exhibited a precursor-product relationship in pulse-chase experiments and appeared to be related by peptide mapping. However, addition of the protease inhibitors tolylsulfonyl-lysyl chloromethylketone (TLCK) and tolylsulfonyl-phenylalanyl chloromethylketone to infected cells during labeling enhanced the recovery of VP 41 and correspondingly decreased VP 38. A similar effect was obtained if addition of TLCK was delayed until the time of cell lysis. Taken together, these observations suggest that during the course of infection, the nucleocapsid polypeptide VP 41 undergoes a transition from a protease-sensitive form (detected by the occurrence of VP 38) to a protease-resistant form. Two other hitherto unreported post-translational modifications of RS virus-induced polypeptides, sulfation and phosphorylation, are described.     

Identification of polypeptide components of adenovirus type 12 tumor antigen from productively and abortively infected cells and from tumor cells

Adenovirus type 12 (Ad12) tumor antigen (T antigen) induced in productively infected KB cells, abortively infected rabbit RK13 cells, and hamster tumor cells (THA cells) was studied by immunoprecipitation and polyacrylamide gel electrophoresis. [³⁵S]Methionine-labeled cell extracts were precipitated with anti-T serum, obtained from hamsters bearing tumors induced by Ad12, and with anti-P serum, obtained from rabbits immunized against Ad12 early proteins induced in RK13 cells. Two polypeptides with 31,000 (31K) and 19K molecular weights are precipitated from all cells. Polypeptides with 16K, 15K, 13K, and 10.5K molecular weights are observed in extracts from KB and THA cells only. T antigen from tumor cells also contains 57K, 50K, 45K, 41K, and 34K polypeptides. Polypeptides with 39K, 28K, and 25K molecular weights are synthesized in KB and RK13 cells, mainly when the cells are pretreated with cycloheximide, but are never seen in extracts from tumor cells. Anti-P serum precipitates the 39K, 31K, 28K, 25K, and 19K polypeptides and also a 60K polypeptide from KB and RK13 cells, which probably corresponds to the DNA-binding protein.     

Replication complexes associated with the morphogenesis of rubella virus

Summary Thin section electron microscopy was used to investigate cellular changes associated with the replication of rubella virus (RV) in Vero cells and to compare these changes to those of the related alphavirus, Semliki Forest virus (SFV). Conspicuous membrane-bound cytoplasmic vacuoles analogous to the alphavirus replication complexes were observed in RV infected cells but not in mock infected cells. The vacuoles were characterised by membrane-bound vesicles measuring about 60 nm which often displayed an irregular dense core and/or a network of fibres. These vesicles were morphologically distinct from RV particles and were generally located at regular intervals on the inner side of the surrounding membrane of the RV replication complex. Degenerating cellular material was often found in the membrane-bound vacuole of a replication complex. The replication complexes were intimately associated with the rough endoplasmic reticulum (RER), which was localised 45–75 nm from the surrounding membrane of the replication complex. Parallel studies of replication complexes in SFV infected cells did not reveal such an intimate association with the RER. RV replication complexes appeared as early as 8 h post infection (p.i.), before detection of RV particles by electron microscopy, and their peak production at 24 h p.i. coincided with the time of maximum virus titre.     

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.     

Isolation and characterization of 'dense particles' from poliovirus-infected HeLa cells.

Dense poliovirus particles (buoyant density 1.44 g/ml in CsCl) isolated from infected HeLa cells contain the normal four structural polypeptides VP1 to VP4, and 35S poliovirus RNA. In addition, small amounts of VPo and single-stranded RNA sedimenting slower than the poliovirus genome are present. Dense particles have a low specific infectivity, are neutralized by type-specific poliovirus antisera, and are detected during growth as early as normal virus but disappear when virus production stops. They appear to represent a different, more open, conformation of the normal virus capsid.     

Rubella virus: intracellular polypeptide synthesis.

Rubella virus (RV) grown in RK13 cells induces a range of polypeptides ranging in molecular weights from 109K to 28K. As RV does not inhibit host metabolism, detection of these requires the use of elevated concentrations of NaCL in the labelling medium to selectively inhibit cellular translation. Several of the viral polypeptides, including p109, p92, and p72 are transient high molecular weight species which may represent precursors of functional viral polypeptides. Only five or six of the polypeptides are synthesized in large amounts at late stages of infection and these probably include the structural components.     

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [³⁵S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1a and b), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.     

Identification of shared antigenic determinants of different polypeptides from Davis and Towne cytomegalovirus strains with monoclonal antibodies

Six colonies producing antibodies were obtained by fusing mouse myeloma SP2-O cells with spleen cells from mice immunized with cytomegalovirus Davis strain. Among 88 surviving clones, 29 produced antibodies detectable by immunofluorescence on infected MRC5 cells and 2 others produced neutralizing antibodies against a homologous virus. Supernatants from these 31 positive clones and 4 others which were negative in immunofluorescence or neutralization were tested for their capacity to bind polypeptides from labelled Davis-infected cell extracts. Only 8 were found to be positive: five clones (A8, E3, F2, F8 and F20) precipitated 76 K, 60 K and 54 K bands; three others (A4, B9 and C24) precipitated 76 K and 54 K only. Surprisingly, these 8 monoclonal antibodies recognized a unique polypeptide 67 K of Towne-infected MRC5 cells. No correlation was found between (1) the pattern of fluorescence on MRC5 cells infected with Davis strain, (2) the neutralizing activity, and (3) the polypeptides recognized by the monoclonal antibodies.     

Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: Synthesis,identification, and processing

The synthesis of virus-specific polypeptides in JLS-V9 and JLS-V5 cells infected with Rauscher leukemia virus (R-MuLV) was studied in pulse-chase experiments, followed by radioimmunoprecipitation analysis with polyvalent and monospecific antisera against R-MuLV proteins. Two glycosylated polypeptides with molecular weights of about 8 (env-pr82) were identified as precursors of the virion envelope polypeptides gp69/71 and p15(E). On the other hand, virion polypeptides p30 and pl5 are derived from a 75,000-(gag-pr75) and a 65,000-dalton (gag-pr65) precursor polypeptide. These precursor-product relations were confirmed by analysis of chymotryptic digests of virion polypeptides and their precursors. In the presence of the arginine analog canavanine two polypeptides with molecular weights of 82,000 and 72,000 (gag-pr82 and gag-pr72, respectively) were synthesized instead of gag-pr75 and gag-pr65. Processing of precursor polypeptides is reduced in the presence of canavanine. From these results, we conclude that gag-pr82 is possibly the primary gag-gene product and is cleaved into gag-pr75. These studies provided the following additional information: First, we established that immediately after cleavage of their precursors, gp69/71 is found on the outer surface of the cell and p30, p15, and p12 leave the cell as components of budding virions. Therefore, these polypeptides were detected intracellularly in very small amounts only. Polypeptide p15(E) was present within the cell as well as on its outer surface. Second, despite a great similarity in virus-specific (precursor) polypeptides detected in JLS-V9 and JLS-V5 cells, small differences in molecular weights of some of these polypeptides were observed after SDS-PAGE.     

Detection of three polypeptides in preparations of bovine viral diarrhoea virus

Summary Radiolabelled bovine viral diarrhoea/mucosal disease virus (BVDV) strains NADL and Oregon C24V were purified by different steps. Following immuno-precipitation, electrophoresis in SDS-polyacrylamide gels revealed three BVDV structural polypeptides with molecular weights of 57 (VP1), 44 (VP2), and 34 (VP3) kd. The two larger BVDV polypeptides VP1 and VP2 were found to be glycosylated (gp57, gp44). The data obtained on BVDV structural proteins demonstrate common features with hog cholera virus and indicate a common grouping with the family Togaviridae.With 2 Figures     

Identification of serotype ii infectious bursal disease virus proteins

Polyacrylamide gel electrophoresis (PAGE) of serotype II IBDV (OH and MO strains) purified from infected Vero cells resolved a previously undetected major viral polypeptide, VP2. The molecular weight (MW) of VP2 was different between the two strains of serotype II. It was 43.5 kDa in strain OH and 44 kDa in strain MO. This was higher than the MW of VP2 in SAL strain of serotype I IBDV which was 41 kDa. VPX (50 kDa), VP3 (33 kDa) and VP4 (30.5 kDa) were similar in both serotype II virus strains but were also of higher MW than VPX (48 kDa), VP3 (32 kDa) and VP4 (30 kDa) of SAL virus. VP1 (80 kDa) had the same MW in both serotypes.     

IgE anti-Borrelia burgdorferi components (p18, p31, p34, p41, p45, p60) and increased blood CD8+CD60+ T cells in children with Lyme disease

Immunoglobulin (Ig) E may provide immunity against Borrelia burgdorferi infection (Lyme disease) in children which lasts throughout adulthood. We investigated the presence and persistence of IgE anti-B. burgdorferi antibodies (Abs) in paediatric patients infected with Lyme disease over time. Serum immunoglobulin levels, presence of IgG and IgE anti-B. burgdorferi components, and distributions of blood T, B and natural killer lymphocyte subsets were studied in B. burgdorferi-infected and -uninfected children (nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay, Western blot, flow cytometry). Total serum IgM, IgG, IgE and IgA levels, and distributions of blood lymphocytes (CD4(+), CD8(+), CD19(+)) of both groups, excluding CD8(+)CD60(+) T cells, were within normal ranges. However, infected, but not uninfected children made IgG anti-B. burgdorferi proteins p18, p31, p34, p41, p45, but not IgG anti-p60, and IgE anti-B. burgdorferi proteins p31, p34, p41, p45, p60, but not IgE anti-p18. These proteins were also detected in an infected child 1 year post-infection. Interestingly, CD8(+)CD60(+) T-cell numbers were significantly increased (fourfold) in infected, compared with uninfected, patients (P=0.001). These results demonstrate that specific IgE anti-B. burgdorferi Abs are generated and persist in children with Lyme disease and that CD8(+)CD60(+) T cells may play an important role in these responses.