Construction and characterization of complementary DNA libraries from Vero cells infected with measles virus

Several cDNA libraries have been generated from poly(A)RNA from Vero cells infected for 24 hours with measles virus. Different protocols for cDNA library construction were compared and some critical steps were evaluated. From these libraries, a measles virus specific sequence corresponding to 885 of 1600 nucleotides of the measles virus phosphoprotein gene has been cloned. The phosphoprotein gene accounts for 1% of the total cDNA library after 24 hours of infection at 37 degrees C. The technique of differential colony hybridization was used to analyze the distribution and change of the poly(A)-RNA expression in uninfected Vero cells and in cells infected with measles virus for 24 hours.     

Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells

Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17-18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29-1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200-300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).     

Isolation of measles virus polypeptides from infected brain tissue by affinity chromatography

A method has been developed to isolate measles virus proteins from infected hamster brain tissue. Suckling hamsters inoculated intracerebrally with the HBS strain of measles virus were used in these studies. Viral proteins were isolated from infected brain lysates by affinity chromatography on Sepharose beads coupled with IgG from rabbit hyperimmune anti-measles serum. The eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred onto blotting matrix, and immunolabelled with anti-measles antibodies. Individual viral proteins were identified by labelling with monoclonal or monospecific antibodies. All viral proteins except the fusion (F1) protein were identifiable on the immunoblots in relative amounts comparable to purified virions. In addition, a second phosphoprotein (P) band not found in purified virions was present in infected brains and cell cultures infected with HBS or LEC strains of virus. This method should be useful for isolating small quantities of viral proteins from large amounts of tissue, and should make possible the characterization of measles virus proteins in persistently infected CNS tissue.     

Induction of measles virus-specific human cytotoxic T cells by purified measles virus nucleocapsid and hemagglutinin polypeptides

The measles virus polypeptide specificity of human measles virus-specific, HLA class II restricted cytotoxic T cells have been examined. Measles virus-specific CTL have been generated using purified measles virus nucleocapsid and hemagglutinin polypeptides during a primary, in vitro stimulation of bulk cultures. Both the purified preparations of measles virus nucleocapsid and hemagglutinin polypeptides were effective in stimulating a measles virus-specific CTL response. The measles virus nucleocapsid-induced CTL response could be blocked by an anti-HLA class II monoclonal antibody but not an anti-HLA class I antiserum. Moreover, considerably less measles virus nucleocapsid was required to stimulate a comparable CTL response than the measles virus hemagglutinin which suggests that the CTL response to measles virus may be skewed towards internal viral determinants of measles virus. These studies indicate that both internal and external components of measles virus are effective in inducing measles virus-specific CTL. The recognition of internal viral components may represent an important part of the T cell mediated immune response to viruses.     

Temperature-sensitive mutants of measles virus produced from persistently infected HeLa cells

Summary A persistent infection with the Edmonston strain of measles virus was established in HeLa cells in the absence of measles virus antibody (HeLa_PI cells). By hemadsorption or immunofluorescence virtually 100 per cent of the cells possessed measles virus components. HeLa_PI cells produced no interferon and were not resistant to superinfection with Newcastle disease virus. HeLa_PI cells contained both smooth (15–18 nm) and rough (20–35 nm) nucleocapsids as detected by electron microscopy. The virus produced from the HeLa_PI cells (MV_PI) varied in titer between 1.5 × 10² and 5.5 × 10⁴ PFU/ml, had a smaller plaque size and was more heat resistant than wild-type measles virus. MV_PI was also found to be temperature-sensitive. The temperature-sensitivity of MV_PI was determined by the efficiency of plaquing at 33° and 39° C in Vero cell monolayers. When HeLa_PI cells were incubated at 33° C, there was a 50-fold increase in virus production as well as a slight increase in the percentage of cells forming infectious centers compared to HeLa_PI cells grown at 37° C. MV_PI readily established a persistent infection in HeLa cells which also released temperature-sensitive virus.With 2 Figures     

Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells

Summary We have shown that a latent infection of herpes simplex virus type 2 (HSV-2) can be established in a human neuroblastoma cell line IMR-32 if the infected cells are cultured at 40°C. In the present study, viral polypeptides and cellular heat-shock proteins which were synthesized in HSV-2 infected IMR-32 cells cultured at 40°C were analyzed by polyacrylamide gel electrophoresis. It was found that the synthesis of late viral polypeptide ICP 5 was markedly reduced in the infected cells at 40°C as compared with those at 37°C. Although infection of IMR-32 cells with HSV-2 at 40°C resulted in shutoff of cellular protein synthesis, it was found that some cellular heat-shock proteins (90, 72 and 70 kd polypeptides) were synthesized and accumulated intracellularly. These findings suggest that modification of cascade regulation of HSV-2 polypeptide synthesis and/or accumulation of heat-shock proteins may be involved in the incomplete arrest of virus growth and in survival of the infected cells, leading to the establishment of HSV-2 latency in IMR-32 cells.     

Unfragmented nucleocapsid of influenza virus in infected cells]

In the cytoplasms of chick embryo fibroblast and Ehrlich ascitic carcinoma cells infected with influenza virus (fowl plague virus), in addition to fragmented virus nucleocapsid larger nucleocapsid structures were found which sedimented in the region of 90 -120S. The structures were detected upon short 3H-uridine label of the cells. Their buoyant density in cesium chloride was higher than that of the fragmented nucleocapsid (1.34 -1.39 g/cm3). In electron microscope, the structures were visualized as thin nonhelical filaments 3.5 nm in diameter, their morphology being no different from that of a similar rapidly sedimenting structure isolated from the nucleoplasms of the same cells. To determine the possibility of transfer of the rapidly sedimenting structure from the nucleus into the cytoplasm, a cell-free system was used containing nuclei from influenza virus-infected cells labeled with 3H-uridine for 5 min, as well as the cytoplasm from uninfected and unlabeled cells. The presence of a labeled rapidly sedimenting structure in the cytoplasm of the cell-free system suggests that the structure is synthesized in the nucleus and then transported into the cytoplasm. The relation of this structure to the fragmented nucleocapsid is unknown. It may be assumed to be its intracellular precursor.     

Cloning of human T cells specific for measles virus haemagglutinin and nucleocapsid.

T cell lines specific for measles virus (MV) were generated from blood of two DR1/DR2 heterozygous healthy donors with a history of past measles infection. The antigenic specificity of 66 T cell clones derived from the lines was studied in a blastogenic assay using whole measles virus and two purified virus components, haemagglutinin and nucleocapsid. Thirty-nine of the clones were specific for one of the two purified antigens. None of the seven synthetic peptides covering 20% of the MV haemagglutinin amino acid sequence stimulated T cell clones with haemagglutinin specificity. Responsiveness of the majority of the clones were restricted by HLA-D/DR antigens, although two clones were isolated that responded only to MV antigens presented by autologous cells. Ten of 11 clones recognizing the nucleocapsid antigen were DR1-restricted, while the haemagglutinin antigen and whole measles virions were recognized more often in association with the DR2 antigen. These results indicate that much of the MV-specific memory T cell response is specific for the haemagglutinin and nucleocapsid virus antigens, with the DR antigen being the main restriction element involved.     

Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus

Three types of virus particles have been recovered from the culture medium of human foreskin fibroblasts infected with human strains of cytomegalovirus (HCMV). Two of these, virions and dense bodies, are routinely observed and have been described by others. The third, produced in lesser amounts, has not been previously characterized. This particle, separable from virions by rate-velocity sedimentation, is morphologically distinguished from them only by core structure. Radiolabeling and biological assays have established that these particles, like dense bodies, lack DNA and are not infectious. Based on these properties, we have designated this virion-like structure as a noninfectious enveloped particle (NIEP). Comparisons of the protein constituents of these three particles has shown that dense bodies have the simplest composition. Approximately 95% of their protein mass is represented by a 69,000 Da (69K) matrix-like protein. While dense bodies appear to have a normal complement of virion glycoproteins, they completely lack other predominant virion species. The protein compositions of virions and NIEPs are more complex than that of dense bodies, and are distinguished from one another by the presence in NIEPs of a 35,000 Da (35K) protein absent from the two other particles. Biosynthetic radiolabeling and cell fractionation experiments have demonstrated that this 35K protein is produced only in infected cells, is phosphorylated and partitions with the nuclear fraction. These and other results suggest that this protein is the HCMV counterpart of the previously described B-capsid proteins VP22a of herpes simplex and 37K of CMV (strain Colburn). NIEPs are produced by all HCMV strains examined and have not been observed in preparations of herpes simplex virus- or Old World monkey CMV-infected cells. Although this particle is generally present in much lower amounts than virions, strain AD169 overproduces NIEPs by approximately 10-fold. We have also found that the additional NIEP protein of AD169 has an apparently larger size (i.e., 36K) than the corresponding protein of other strains. The correlation between AD169 NIEP overproduction and its altered protein suggests that the two may be causally related.     

Biosynthesis of measles virus hemagglutinin in persistently infected cells

The synthesis of the hemagglutinin (HA) glycoprotein of measles virus was investigated in a persistently infected cell line using a monoclonal anti-HA. The synthesis of the HA protein was shown to be associated with the rough endoplasmic reticulum. The unglycosylated (HA0) apoprotein is synthesized as a 65,000 dalton peptide and is inserted into the rough endoplasmic reticulum as a transmembrane protein with approximately 2 to 3000 daltons of the peptide exposed to the cytoplasmic membrane surface. Primary glycosylation of the HA protein was found to occur through the lipid-linked carrier, dolichol-phosphate, as determined by inhibition of glycosylation by tunicamycin. Glycosylation, however, was not a prerequisite for membrane insertion. Endo-beta-N-acetylglucosaminidase H digestion of the fully glycosylated HA protein indicated that both simple and complex oligosaccharides are present on the surface glycoprotein.     

Isolation and characterization of actin from cultured BHK cells

Summary Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg²⁺ ions (0.2mm CaCl₂, 85mm KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca²⁺ ions for BHK actin than for muscle actin (12mm and 8mm respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.     

Isolation and characterization of gelsolin from cultured BHK cells

Summary The Ca²⁺-dependent actin-polymerization nucleating protein of the cytoplasmic fraction of Baby hamster kidney (BHK) C13 cells has been isolated by anion-exchange, hydroxyapatite and gel-filtration chromatography. This protein has been identified as a cytoplasmic gelsolin by the following criteria: molecular mass of 90 kDa on SDS-PAGE, immunocrossreactivity with pig plasma gelsolin and similar actin-binding properties to gelsolins purified from other sources. BHK gelsolin forms a 12 ternary complex with rabbit muscle actin that is dependent on the presence of Ca²⁺. The ternary complex is dissociated on chelation of Ca²⁺ with EGTA to a binary complex and free actin. BHK gelsolin nucleates the polymerization of pyrene-labelled G-actin in a Ca²⁺-dependent manner. The proportion of unpolymerized monomer is increased in the presence of BHK gelsolin by an amount consistent with capping of the positive filament ends. The rate of actin depolymerization induced by diluting F-actin to below its critical concentration (C_c) is unaffected by the presence of BHK gelsolin in EGTA. However, in the presence of Ca²⁺ the rate of depolymerization is increased indicating that BHK gelsolin severs actin filaments in a Ca²⁺-dependent manner.     

Isolation and characterization of double stranded RNA containing infectious viral genome RNA from cells infected with Semliki Forest virus

Summary A double stranded virus specific RNA sedimenting at about 19S on sucrose density gradients has been isolated from BHK-21 cells infected with Semliki Forest virus (SFV). The molecule consists of double stranded RNA (ds RNA) since it is labeled with³H-uridine, is souble in 2m LiCl, resistant against treatment with DNase and RNase at 2×SSC, hydrolyzed by alkali treatment, has a sharp thermal melting point at 89° in 1/10 SSC, and an extended appearance under non denaturing conditions in the electronmicroscope. The following findings show that it consists of intact, infectious 42S RNA similar or identical to the genome RNA of SFV complexed to a complementary 42S minus strand RNA: 1. Denaturation converts the ds RNA into molecules cosedimenting with 42S RNA isolated from SFV particles. 2. About 50 per cent of the radioactivity of³H-uridine labeled 42S RNA molecules generated from 19S ds RNA by denaturation hybridizes to 42S viral RNA. 3. The specific infectivity of denatured 19S ds RNA is about half of that of similarly treated viral 42S RNA. Further properties of this molecule are discussed.With 5 Figures     

Isolation and characterization of the nonglycosylated membrane protein and a nucleocapsid complex from the paramyxovirus SV5.

The smallest polypeptide of the paramyxovirus SV5 (simian virus 5) the M protein, which is the major nonglycosylated protein of the viral envelope was isolated. The procedure used consisted of solubilization of the glycoproteins with Triton X-100, leaving an insoluble aggregate consisting of the protein subunit of the nucleocapsid (NP), the M protein, and a protein with a molecular weight of ～50,000 (protein 5). Further treatment of this aggregate with 1% deoxycholate and 0.5 M urea followed by centrifugation on a discontinuous sucrose-on-cesium chloride gradient separated the aggregate into a fraction at the sucrose-cesium chloride interface consisting of the M polypeptide and a complex banding in 30% cesium chloride consisting of the nucleocapsid and protein 5. This technique yielded large quantities of highly purified M protein and a nucleocapsid complex that were amenable to biological and chemical analysis. Amino acid analysis of the purified membrane protein revealed a chemical composition of 64% neutral amino acids. The finding of protein 5 with the nucleocapsid after isolation on the cesium chloride gradient suggests that this protein may be associated with the nucleocapsid in the virion.     

Isolation and characterization of interferon-producing reticuloendothelial cells from mouse epidermis

A homogeneous population of trypsin-resistant epidermal cells has been isolated from newborn ICR mice. These cells are characterized by adherence, receptors for Fc-IgG, ATPase activity, phagocytosis of latex particles and opsonized sheep erythrocytes, and secretion of lysozyme and interferon. The production of interferon by these cells suggests that they may be important in protection against viral infections of the skin as well as in regulation of immune responses. The ultrastructure of these trypsin-resistant epidermal cells shows striking similarity to that of reticuloendothelial cells.     

Isolation and characterization of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi

A new procedure has been developed which allows the concomitant isolation of viable parasites and host cell plasma membranes from erythrocytes infected with Plasmodium chabaudi trophozoites. The average final yield of parasites is 56%. Free parasites reveal a well preserved ultrastructure, incorporate [14C]isoleucine for at least 3 h, and synthesize about the same proteins as parasites within erythrocytes as monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-autoradiography. The host cell plasma membranes can be isolated in the form of ghosts with an average yield of 27%. The ghosts possess a structurally intact plasma membrane as revealed by freeze-etch electron microscopy. The ghosts are regularly associated with seven neo-proteins as identified by SDS-PAGE and isoelectric focusing (IEF)/SDS-PAGE. These neo-proteins have the following apparent molecular masses: 154 kDa, 145 kDa, 90 kDa, 72 kDa (pI 4.5), 67 kDa, 52 kDa, and 33 kDa (pI 5.7), respectively. The contamination of ghosts by parasite material and, conversely, the contamination of parasites by host cell plasma membranes is very low as demonstrated by light and electron microscopy, lactoperoxidase-mediated radioiodination and the distribution of the typical parasite marker enzymes such as choline kinase, cholinephosphotransferase and ethanolaminephosphotransferase.