Changes in liver and L-cell plasma membranes during infection with Coxiella burnetii.

Changes in plasma membrane proteins of guinea pig liver and L-929 cells were studied during infection with Coxiella burnetii. Polypeptide species resolved by disc polyacrylamide gel electrophoresis with sodium dodecyl sulfate showed quantitative but no qualitative differences between uninfected and infected samples. When the O'Farrell technique of isoelectric focusing, followed by sodium dodecyl sulfate-slab gel polyacrylamide gel electrophoresis, was employed, additional polypeptides were resolved. Livers and L cells were labeled with [3H]-glucosamine. Infected livers incorporated less [3H]glucosamine in the membrane proteins than uninfected material, presumably indicating lower glycoprotein levels. Infected L-cell membranes incorporated greater amounts of [3H]glucosamine, and also were labeled to a greater extent than uninfected membranes, employing the [125I]lactoperoxidase technique. Uninfected L cells showed a greater agglutinability with concanavalin A than did infected cells. Infected livers had much greater levels of cyclic adenosine 3',5'-monophosphate. The data indicate changes in plasma membranes as a result of infection. Possible physiological consequences of membrane changes are discussed.     

DNA probes detect genomic diversity in Theileria parva stocks

Different stocks of Theileria parva were analysed for restriction fragment length polymorphisms by agarose gel electrophoresis, orthogonal-field-alternation gel electrophoresis (OFAGE) and Southern hybridization with DNA probes. Polymorphisms seen with DNA from purified piroplasms of different T. parva stocks, after digestion with restriction enzymes, were more clearly apparent with OFAGE than with standard agarose gel electrophoresis. Genomic differences between these theilerial parasites were investigated further using three DNA probes, which were selected from a genomic library of T. parva (Muguga) piroplasm DNA cloned in lambda gt11. All three clones hybridized to T. parva DNA in preparations from schizont-infected bovine lymphoblastoid cells and to DNA from intraerythrocytic piroplasms. These probes did not, however, hybridize under high stringency conditions to DNA prepared from uninfected bovine lymphoblasts, T. mutans piroplasms, or bovine lymphoblasts infected with T. annulata or T. taurotragi. The five Kenyan stocks of T. parva that were tested showed characteristic hybridization patterns with these DNA probes. Our results show that DNA probes can be used to distinguish selected stocks of T. parva by hybridization to DNA either from intraerythrocytic piroplasms taken from infected cattle, or from isolates of schizont-infected bovine lymphoblastoid cells that are maintained continuously in vitro.     

Herpes simplex virus type 1 infection of endothelium reduces collagen and fibronectin synthesis

Protein synthesis was assessed in virus-infected and -uninfected cultures of bovine aorta endothelial cells by following the incorporation of [14C]proline into nondialyzable protein and by use of an ELISA assay. Monolayers were infected at confluency by incubating with herpes simplex virus type I at a multiplicity of infection (ratio of virus to cells) of 1.0, 1 hour prior to the introduction of the [14C]proline. Samples were taken from infected and uninfected cultures at various time points postinfection from both medium and cell layer fractions and analyzed for total 14C-labeled protein, 14C-labeled collagen, and 14C-labeled fibronectin. Medium proteins were analyzed by polyacrylamide gel electrophoresis, gel filtration, and electroimmunoblotting. No change was detected in total [14C] incorporation into nondialyzable protein in infected compared with uninfected endothelial cells; however, there was a significant decrease in the synthesis of collagen and fibronectin in infected cultures. The hydroxy[14C]proline content of fractionated medium proteins showed no significant differences between infected and uninfected cultures; therefore, the decrease in collagen synthesis cannot be explained by increased collagen degradation. These data suggest that infection of bovine endothelial cells causes a reduction in the synthesis of collagen and fibronectin.     

Immune responses of cattle to Theileria parva (East Coast fever): specificity of cytotoxic cells generated in vivo and in vitro.

Examination of the specificity of cytotoxicity generated in vitro and in vivo against infected bovine lymphoblasts revealed that cytotoxic T lymphocytes (CTL) obtained from cattle immune to Theileria parva recognized parasite-induced alterations associated with major histocompatibility complex (MHC) antigens on the membrane of infected autologous cells. By comparison, cytotoxicity generated in vitro in an autologous Theilerial-lymphocyte culture (AuTLC) contained both CTL and activity akin to that of natural-killer (NK) cells. The addition to the AuTLC of 2 inhibitors of glycosylation, tunicamycin (Tun) and 2-desoxy-D-glucose (2-DOG) abolished both the proliferative response and the generation of cytotoxicity. While the addition of Tun or 2-DOG in conventional cell-mediated lympholysis (CML) assays did not modify the effector function of cytotoxic cells, pretreatment of target cells with either compound prevented lysis by CTL, but not by NK cells. Although parasite-induced antigens have not been purified from infected bovine lymphoblasts, the present study indicated that these are likely to be glycoprotein or carbohydrate in character, and that their recognition on autologous cells is a consistent feature of CTL from immune cattle.     

Resolution of two surface glycoproteins from human parainfluenza-3 virus by crossed immunoelectrophoresis

The technique of two-dimensional crossed immunoelectrophoresis (CIE) was used to resolve two glycoproteins from purified human parainfluenza type 3 virus. Virus preparations were extracted with Triton X-100 and fractionated by centrifugation in a Beckman airfuge. Two immunoprecipitates were detected by CIE in the supernatant fractions, but were not found in the pellets from extracted virus. Viral glycoproteins labeled with [35S]methionine were isolated by affinity chromatography on concanavalin A (Con A) agarose columns, resolved by CIE and detected by autoradiography. Resolution of two glycoprotein peaks from as little as 4.5 micrograms of protein from extracted virus is consistent with results from polyacrylamide gel patterns showing two unique glycoproteins with molecular weights of 48 kd and 65 kd.     

Phenotypic characterization ofTheileria parva schizonts by two-dimensional gel electrophoresis

Biosynthetically radiolabelledTheileria parva schizonts were purified from bovine lymphoblastoid cells and their proteins were analyzed by two-dimensional gel electrophoresis and autoradiography. The protein spot patterns of schizont proteins from three stocks ofT. parva parva indicated that the phenotypic diversity among the stocks was minimal, with the Mariakani and Uganda stocks being identical and the Muguga stock showing only a few differences in minor spots. Comparison of the spot patterns of schizonts of threeT. parva subspecies showed thatT. p. parva andT. p. bovis differed in only one protein and thus could not be reliably distinguished on the basis of their protein differences. However,T. p. lawrencei showed several protein differences and could be distinguished easily from the other subspecies. Differences in schizont-protein spot patterns were also seen when two different cell lines were infected with the sameTheileria stabilate, when one cell line was infected with two different stabilates of the same stock and when uncloned and cloned infected cell lines were used. These results suggest the possibility that selection of phenotypically different parasites could occur in vivo or in vitro.II RAD publication 641     

Channel catfish virus gene 50 encodes a secreted, mucin-like glycoprotein

Cells infected with the wild-type (WT) strain of channel catfish virus (CCV) secreted a glycoprotein with an apparent molecular mass (MM) superior to 200 kDa into the culture medium. This protein, designated gp250, was the sole viral glycoprotein detected in the culture medium after [3H]mannose labeling of the infected cells. When cells were infected with the attenuated V60 strain, a glycoprotein of 135 kDa (designated gp135) was detected instead of gp250. Because WT gene 50 is predicted to encode a secreted, mucin-type glycoprotein, we expressed this gene transiently and detected a glycoprotein of the same apparent MM as gp250 in the culture medium of transfected catfish cells. The increased mobility in SDS-PAGE of the secreted V60 glycoprotein correlated with the presence of a major deletion in V60 gene 50. Therefore, we concluded that gp250 in the WT and gp135 in the V60 strains are both likely encoded by gene 50. An important shift in the relative mobility of gp250 in SDS-PAGE was observed after tunicamycin treatment of infected cells labeled with [3H]glucosamine, confirming the presence of N-linked sugars on gp250. We observed variations in the size of PCR products derived from gene 50 amplification in three different field isolates. Such genetic variations are a characteristic feature of mucin genes and are linked to crossing-over events between internal repeated sequences, such as those present in gene 50.     

The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication.

The ability of vaccinia virus to inhibit processes of cap-dependent translational initiation by inactivating the eukaryotic translation initiation factor 4E (eIF-4E) has been examined. Analyses of the quantities of eIF-4E present in either uninfected mouse L929 cells or vaccinia virus-infected cells showed that during the first 12 hr of virus replication, when there is a marked decrease in host gene expression in infected cells, there is no change in the total amount of eIF-4E present. Analyses of eIF-4E that was metabolically labeled with [32P] and then purified by affinity chromatography using m7GTP-Sepharose 4B, indicated that neither the incorporation of radiolabel into eIF-4E nor the amounts of eIF-4E capable of binding to cap structures changed significantly during virus replication. Immunodetection of phosphorylated and unphosphorylated eIF-4E in cell lysates fractionated by two-dimensional gel electrophoresis showed that the steady-state levels of phosphorylated and unphosphorylated forms of eIF-4E were similar in uninfected and virus-infected cells. These results suggest that vaccinia virus does not gain preferential translation of viral mRNAs over other mRNAs in the cell by reducing either eIF-4E phosphorylation or its ability to bind to the cap structure.     

Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i-RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis.     

Studies on glycopeptides of herpes simplex virus infected cells

Summary Trypsinates from HSV infected cells, radioactively labeled with glucosamine and galactose were further digested by pronase. The digests were subjected to gel filtration, and it was shown that some of the eluted material consisted of saccharides, highly devoid of residual peptide. This material eluted between linear dextran markers of 19,000 and 3000 daltons. Only slight differences between uninfected and HSV infected cells in galactose and glucosamine derived radioactivity profiles of the chromatograms were detected. High molecular weight glucosamine labeled material, present in digest from uninfected cells, was not detectable in digests from HSV infected cells.The concanavalin A adsorbability of eluted fractions was tested. Changes in the relative adsorbability between materials from HSV and uninfected cells were present, especially for the glucosamine labeled saccharides.With 6 Figures     

Analysis of a glycoprotein of human herpesvirus 6 (HHV-6) using monoclonal antibodies

The virus-specific polypeptides of human herpesvirus 6 (HHV-6) were studied. Six hybridoma clones secreting monoclonal antibodies were established. Localized cytoplasmic antigen and surface antigens were found in and on the infected cells, respectively, by using the immunofluorescent test with those antibodies. In the neutralization test, two clones (OHV3 and OHV9) neutralized HHV-6, but the other five monoclonal antibodies did not. When infected cells were labeled with [35S]methionine and the antigens were immunoprecipitated by OHV3 and OHV9 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two polypeptides with molecular weights of 98,000 (98K) and 92K were detected. Furthermore, it was found that these polypeptides were glycosylated, because they were labeled with [14C]mannose. Pulse-chase studies revealed that precursor molecules were cotranslationaly glycosylated to produce 98K glycoprotein and they were replaced by 92K glycoprotein. Endo-beta-N-acetylglucosaminidases H and F reduced those glycoproteins to putative precursor molecules of 75 kDa. This indicates that N-linked high-mannose sugars associate to 98K and 92K glycoproteins.     

A simple and efficient method for purifying and quantifying schizonts fromTheileria parva-infected cells

An improved method for the purification ofTheileria parva schizonts from infected bovine cells is described. The technique is simpler and more rapid than previously described methods and gives rise to greater yields of schizonts with negligible contamination by host-cell components. In addition, a fluorescent staining technique was developed whereby live schizonts purified from infected cells can be enumerated and sorted using the flow cytometer. An assessment of the quality of schizonts prepared according to our method as a source of RNA for the construction of parasite cDNA libraries suggests that RNA derived from these preparations is free of host nucleic acids.     

Effect of herpes simplex virus type 1 on cellular pools of oligosaccharide-lipid

Incorporation of [3H]mannose into cellular pools of mannosylphosphoryl dolichol (Man-P-Dol), oligosaccharide-lipid, and glycoprotein was measured and compared in herpes simplex virus type 1 (HSV-1)-infected cells and -uninfected cells. While mannose incorporation into the monosaccharide-dolichol fraction was similar in infected or uninfected Vero cells, incorporation into the oligosaccharide-lipid fraction was markedly reduced in HSV-1-infected cells (64% of control levels). In contrast, mannose incorporation into glycoprotein was significantly increased in virus-infected cells versus uninfected cells (194% of control levels). The kinetics of incorporation into the various fractions was examined and it was determined that there was minimal increase in mannose incorporation into oligosaccharide-lipid after 8 hr postinfection in virus-infected cells. This corresponded to the time at which nonglycosylated precursors of the HSV-1 glycoproteins were first detected in association with the nuclear fraction. These data suggest that there is an accelerated turnover of oligosaccharide-lipid in virus-infected Vero cells which is most likely due to extensive glycoprotein synthesis.     

Comparison of proteins synthesized by two different isolates of Anaplasma marginale.

We present results on the initial definition of proteins synthesized by two isolates of Anaplasma marginale. Bovine erythrocytes infected with A. marginale were radioactively labeled with [35S]methionine or a 3H-amino acid mixture during short-term in vitro culture. The labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed protein bands of various apparent molecular weights from less than 14,000 to greater than 200,000. The bands observed represented A. marginale proteins because (i) uninfected erythrocytes from the same animal did not incorporate radioisotope during identical culture conditions, and (ii) the incorporation of radioisotope into proteins during culture of infected erythrocytes was inhibited by tetracycline but not by cycloheximide. The radioactive protein profiles of two different isolates of A. marginale, from Washington and Florida, were compared by two-dimensional gel electrophoresis. About 200 proteins were resolved in each case. Several proteins differed in position when the two-dimensional gel maps were compared, indicating variations in protein structure between the two A. marginale isolates.     

Equine cytomegalovirus: structural proteins of virions and nucleocapsids

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.     

The nuclear matrix is involved in herpes simplex virogenesis

Nuclear matrices have been purified from BHK cells infected with herpes simplex virus type 1 (HSV-1) and compared with nuclear matrices from uninfected BHK cells. Both showed typical structure with residual nucleolus, peripheral lamina with pore complexes, and a nonchromatin fibrillar network. Numerous viral capsids were seen stuck to that framework in matrices from infected cells. SDS-PAGE and fluorography analysis showed that polypeptides normally found in the noninfected BHK nuclear matrix were still present in the infected matrices. Nine additional virus-induced polypeptides were detected 15 hr after infection by labeling with [³⁵S]methionine. This suggests that the nuclear matrix is involved in the formation of the capsid or in the encapsidation process. Twelve DNA-binding proteins were also detected in infected matrices.     

Inhibition of bovine leukemia virus release by antiviral antibodies

Summary Peripheral blood lymphocytes from bovine leukemia virus (BLV) infected cattle were grownin vitro with serums from BLV-infected and uninfected cattle, sheep and rabbits. Only serums from infected animals which had antibody to gp 45/55, the major glycoprotein antigen, inhibited the release of virus from the cells. Although viral antigens could be detected in the cells themselves, none were detected in the supernatants of cultures grown with these serums. Normal serums and serums with antibody only to p23, the internal BLV antigen, did not inhibit virus release. To identify the factor responsible for virus release inhibition, 2 inhibitory serums were absorbed with either gp 45/55, p23 or tissue culture cells from BLV-infected and uninfected cell lines. The results indicated that antibody to gp 45/55 is the factor responsible for virus release inhibition.     

Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum

We have investigated the protein and glycoprotein content of Plasmodium falciparum merozoites by metabolically labeling cultures of schizont-stage parasites with [³⁵S]methionine or with [³H]glucosamine followed by incubation in nonradioactive medium to allow the schizonts to mature into merozoites, infect new erythrocytes, and develop into ring-stage parasites. The ring stages were separated from schizonts by sedimentation through Percoll. Labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Using [³⁵S]methionine, four major proteins (p) with apparent relative molecular weights (M_r) = 202k, 136k, 82k, and 46k and two proteins of intermediate labeling (M_r = 185k and 142k) were observed in the schizont-labeled ring-stage parasites. Because corresponding proteins were also observed in the schizont stage, we conclude that they had been present in the invading merozoite. In contrast, prominent proteins which were generally labeled during the ring stage and some major schizont-stage proteins were virtually absent in the schizont-labeled ring-stage. By labeling the parasite proteins with [³H]glucosamine followed by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five major glycoproteins (gp) of apparent M_r = 185k, 88k, 56k, 46k, and 34k were identified. Their presence in both the schizont and the schizont-labeled ring stage demonstrated that the merozoite contains glycoproteins. Immune owl monkey serum recognized all five glycoproteins. A comparison of proteins by two-dimensional gel electrophoresis (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis) suggested that p185 and gp185 were identical, as were p46 and gp46.     

I. Effect of Trichinella spiralis infection on the migration of mesenteric lymphoblasts and mesenteric T lymphoblasts in syngeneic mice.

The migration of [125I]UdR-labelled mesenteric lymph node cells in NIH strain mice at various times after inis produced an enhanced accumulation of mesenteric immunoblasts in the small intestine at 2 and 4 days after infection but not at later times. The enhanced migration occurred when using cells from both uninfected and infected donors, denoting an absence of antigenic specificity. This effect is not secondary to a reduced arrival of cells at sites away from the gut in infected mice, but to a primary increase of the arrival in the small intestine. Mesenteric T lymphoblasts (separated on a nylon-wool column) migrated to the small intestine of uninfected recipients and appear to be a major portion of the population which migrate to the gut of infected recipients. Our results were confirmed using 51Cr to label mesenteric cells. We conclude that the parasite causes the small intestine to become more attractive or retentive for mesenteric blast cells early during infection.     

Proteins of Rauscher murine leukemia virus: resolution of a 70,000-dalton, Nonglycosylated polypeptide containing p30 peptide sequences.

A viral-specific polypeptide of molecular weight about 70,000 which does not incorporate radioactive glucosamine or fucose and which does not stain positively for carbohydrates is present in preparations of purified Rauscher murine leukemia virus. This polypeptide can be resolved from the major glycoprotein(s) previously designated $\text{gp69}\text{71}$ by high resolution sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The 70,000-dalton polypeptide is present in Rauscher virus-infected cells but not in nonproducer, uninfected N.I.H. Swiss mouse embryo cells. The [³⁵S]methionine-labeled tryptic peptide patterns of the virion and the intracellular 70,000-dalton polypeptide are identical, and each contains [³⁵S]methionine-labeled tryptic peptides characteristic of virion p30, indicating a possible precursor relationship to p30.