Virus-induced Polypeptides Research Articles

Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase.

Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many others proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1- and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.

Bunyamwera virus-induced polypeptide synthesis.

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.

Isatin-beta-thiosemicarbazone causes premature cessation of vaccinia virus-induced late post-replicative polypeptide synthesis.

The effects of isatin-beta-thiosemicarbazone on vaccinia virus-induced polypeptide synthesis has been examined by polyacrylamide gel electrophoresis and autoradiography. The synthesis of pre-replicative and early post-replicative polypeptides proceeded normally in the presence of the drug; the onset of late post-replicative polypeptide synthesis occurred with normal timing under these conditions but the rate of synthesis of all virus polypeptides then declined rapidly.

Chick embryo lethal orphan (CELO) virus-induced early and late polypeptides

Polypeptides of chick embryo lethal orphan virus (an avian adenovirus) were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Virions ( p = 1.338 g/cm 3) and virion-like particles having lower density (light particles, p = 1.289 g/cm 3) were composed of at least 11 and 14 types of polypeptides, respectively. At least 23 virus-induced polypeptides in infected cells (primary monolayer culture of chicken kidney cells) were detected; eight polypeptides (early polypeptides) were synthesized even in the presence of 1-β- d-arabinofuranosyl cytosine, but the others (late polypeptides) were not. On the basis of migration in SDS-PAGE, 5 polypeptides were found to be common to virions and infected cells; 5, to light particles and infected cells; 10, to virions and light particles; and 4, to all three. Scarcely any of the early polypeptides were found in the soluble fraction when extracted with the low salt buffer. Some of the early polypeptides were solubilized with the high salt buffer. Other early polypeptides were solubilized by sodium deoxycholate; three of these were found in the so-called “M-band,” as are early polypeptides of human adenoviruses. An experiment with radioactive glucosamine indicated that one of early polypeptides found in the M-band was a glycoprotein.

Genetic analysis of adenovirus type 2 IV. Coordinate regulation of polypeptides 80K, Illa, and V

An adenovirus type 2 temperature-sensitive mutant, ts3, deficient in virion assembly was studied by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopy. A total of 28 virus-induced polypeptides could be detected, four of which were precursor proteins. At the nonpermissive temperature, ts3 failed to synthesize a newly identified nonstructural polypeptide 80K. Polypeptide V was absent, but it may have been replaced by an arginine-rich polypeptide which was oversynthesized and migrated as a 50K band regardless of temperature. Similarly, polypeptide 55K (= IVa 2) was also oversynthesized and migrated slower than WT-55K (= IVa 2). In addition, several polypeptides were synthesized at increased or decreased levels, compared with WT. Polypeptide IIIa was sometimes resolved into three bands (66K, 67K, 68K) in WT, while ts3-IIIa lacked the 66K and 68K components. Polypeptide 36K was found to be rich in arginine. Consistent with the notion that the processing of PVII into VII requires virion assembly, no processing of ts3-PVII could be observed. By contrast, the cleavage products, VI and VIII, appeared normally, thus disputing the virion assembly requirement previously postulated for these polypeptides. Electron microscopy of ts3-infected cells revealed two hitherto undescribed intranuclear structures, possible tubular forms in addition to normal virions at the permissive temperature, and roughly spherical, core-like structures of 80–100 nm at the restrictive temperature. These results suggest that the pleiotropic effects of the ts3 mutation arrest virus assembly at a corelike structure.

Effect of Zinc Ions on Synthesis of Herpes Simplex Virus Type 2-Induced Polypeptides

Treatment of herpes simplex virus-infected cells with zinc sulfate (ZnSO4) inhibited formation of virus progeny, but had little effect on virus DNA synthesis. Analysis of polypeptides synthesized in infected cells in the presence of ZnSO4 revealed a markedly reduced synthesis of virus-induced polypeptides.

Effect of 5-bromodeoxyuridine on vaccinia virus-induced polypeptide synthesis: selective inhibition of the synthesis of some post-replicative polypeptides.

The effect of 5-bromodeoxyuridine (BrdU) on vaccinia virus-induced polypeptide synthesis in BSC-1 cells has been investigated. Most virus-induced pre- and post-replicative polypeptides were synthesized in concentrations of 5-bromodeoxyuridine that inhibited virus growth. The synthesis of a few post-replicative polypeptides, however, was severely inhibited under these conditions; included in this group was the precursor of a major core component, polypeptide P4b. A delay in the switch-off of pre-replicative polypeptide synthesis and in the onset of post-replicative polypeptide synthesis was also observed. The significance of these observations is discussed.

Genetic analysis of adenovirus type 2 III. Temperature sensitivity of processing viral proteins.

Using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of [35S]methionine-labeled adenovirus type 2-infected KB cell extracts, a total of 23 virus-induced polypeptides was detected. This technique was applied to the analysis of the temperature-sensitive mutant, ts 1, which has previously been shown to be defective in a late function. By means of pulse-chase experiments, ts 1 was shown to be defective in the processing of the precursor polypeptide (Pre VII) to the major core protein VII. Two other putative precursor polypeptides, Va (27K) and Vb (24K), were also not processed. Thus, the ts 1 mutation blocked the appearance of six post-translational clevage products, i. e., polypeptides VI, VII, VIII, X, XI, and XII. All of these polypeptides are virion components. Processing was temperature sensitive in a shift-up experiment, whereas it was normal in a shift-down experiment. The kinetics of the temperature-shift experiments suggested that infectious virus could be recovered if enough time is provided for processing to take place. Processing was not inhibited by cycloheximide. The analysis of purified virus particles and empty shells (TCs) revealed the presence of the precursor and putative precursor polypeptides Pre-VII, Va and Vb, instead of their cleavage products, in both types of particles. Based on these results we propose that the ts 1 gene codes for or regulates an endoprotease which is responsible for the completion of the last step in virus maturation, that is, the conversion of "young virions" into mature infectious virions by a series of maturation cleavages.

Electrophoretic analysis of capsid and non-capsid polypeptides of echovirus 12, and selective inhibtion of the formation of virus particles by actinomycin D.

Electrophoretic analysis of purified echovirus virus particles yielded four polypeptides of mol. wt. 37000, 30000, 25000 and 7600. The 75S empty capsids of echovirus 12 lack the 25000 and 7600 mol. wt. polypeptides, and possess polypeptides of 41000 mol. wt. A total of 14 virus-induced polypeptide species was found in the cytoplasm of infected cells. Actinomycin D reduced the synthesis of virus, virus RNA, and virus polypeptides and also reduced the proportion of virus particles to empty capsids in the virus yields.

Isolation and characterization of a large molecular-weight polypeptide of herpes simplex virus type 1

Infection of human embryonic lung cells at the nonpermissive temperature (39°) with tsB2, a DNA-negative mutant of herpes simplex virus type 1 (HSV-1) resulted in the accumulation of a large molecular-weight (175,000) virus-induced polypeptide detectable by SDS-polyacrylamide gel electrophoresis. This polypeptide (VP175) was detectable only in small amounts and was found mainly in the nuclear fraction of cells infected with tsB2 at the permissive temperature (34°) or with wild-type HSV-1 at both 34° and 39°. However at 39° VP175 accumulated to become the major component in both the cytoplasmic and nuclear fractions of tsB2-infected cells. Identical results were obtained with a second mutant (tsB21) in the same complementation group. Temperature-shift studies suggest that the events responsible for the accumulation of VP175 at 39° occur early in the replicative cycle. With the use of a combination of SDS-preparative and analytical disc gel electrophoresis, VP175 was isolated and rabbit antisera to this polypeptide were prepared. By immunofluorescence assay, anti-VP175 sera reacted only with the nucleus of wild-type HSV-1-infected cells, whereas tsB2-infected cells cultured at 39° showed both cytoplasmic and nuclear immunofluorescence. In addition, the anti-VP175 serum reacted preferentially with HSV-1 as compared with HSV-2-infected cells, suggesting a possible type specificity for the reagent.

Adenovirus polypeptide synthesis in the presence of non-replicating poliovirus.

SUMMARY The effect of superinfection of adenovirus-infected cells with poliovirus in the presence of guanidine (P+G) has been analysed by pulse-labelling of cells with [35S]-methionine followed by polyacrylamide gel electrophoresis and autoradiography. The addition of P+G while reducing total polypeptide synthesis selectively inhibits cellular polypeptide synthesis and allows the visualization, particularly early in infection, of other labelled polypeptides previously obscured. Thus, at least three polypeptides i.e. ICSP-3, core-1 and ‘p-core 2’ can be detected in the absence of virus DNA synthesis. The addition of P+G also causes a retardation of the synthesis of the virus-induced polypeptides giving a clear sequence of translation viz ICSP-3: core-1: p-core 2 → ICSP-1 → hexon → penton base → fibre. Since a similar sequence can be demonstrated by the use of the inhibitor cordycepin, it seems possible that this reflects a sequence of transcription. P+G addition also effectively inhibits the formation of virus particles.

A temperature-sensitive mutant of herpes simplex virus type 1 defective in the synthesis of the major capsid polypeptide.

SUMMARY The virus-induced polypeptides synthesized in human embryonic lung cells infected with ts 4, a DNA negative temperature-sensitive mutant of herpes simplex virus type I, were examined at the permissive (34 °C) and non-permissive (39 °C) temperatures using SDS-polyacrylamide gel electrophoresis. Cells infected with ts 4 at 34 °C synthesized virus-specific polypeptides in the same proportion as did wild-type-infected cells at 34 and 39 °C. Cells infected with ts 4 at 39 °C exhibited multiple polypeptide defects, the most prominent of which was the inhibition of the synthesis of the major virus capsid polypeptide, VP154. The expression of the temperature-sensitive defect relating to VP154 only occurred prior to 4 h after infection, as shown by shift-up studies. The expression of the temperature-sensitive defect at 39 °C relating to VP154 could be reversed by shifting down infected cells to 34 °C, even after 12 h at the non-permissive temperature. This reversal occurred in the presence of cytosine arabinoside but not in the presence of actinomycin D.

Immunochemical procedure for partial purification of newly synthesized proteins in polyoma virus-infected mouse cells.

A broad-spectrum antiserum has been obtained against nuclear proteins from normal mouse fibroblasts. Solid phase immunoadsorbants prepared from the serum specifically bind the corresponding antigens. The removal in this way of a significant fraction of host proteins leads to partial purification of viral and virus-induced polypeptides from polyoma virus-infected cell extracts. Four main peptides can be selected, with respective molecular weights of 90,000, 70,000, 46,000, and 41,000.

The polypeptides of adenovirus: IV. Detection of early and late virus-induced polypeptides and their distribution in subcellular fractions

Three “early” polypeptides induced by adenovirus type 2 infection of HeLa cells have been detected. Two, E1 and E2, are found in cytoplasmic extracts. E3 is present in nuclear extracts. Increased resolution in acrylamide gel electrophoresis and prefractionation permitted these observations along with the detection of “late” polypeptides similar in amount but having higher apparent MW than the core polypeptides of the adenovirion. At late times the great bulk of protein is in structural polypeptides. Late nonstructural proteins cannot be positively identified and, if present, are synthesized at levels of 0.1–1% of the structural proteins.

Processing of adenovirus 2-induced proteins.

Analysis of (35)S-methionine-labeled extracts of adenovirus 2-infected KB cells revealed 22 virus-induced polypeptide components. Most proteins of the virion were easily detected in extracts of whole cells labeled for short periods between 15 and 30 h after infection; however, several virion components were conspicuously absent. Radioactivity appeared in two of these virion components during a chase in nonradioactive medium, and this appearance was paralleled by a decrease in the radioactivity associated with two nonvirion adenovirus-induced proteins, results which imply precursor-product relationships for these components. Comparison of one of the chasable adenovirus-induced components (designated P-VII; mass of 20,000 daltons) and the major core protein (VII; mass of 18,500 daltons) of the virion showed that they have four common methionine-containing tryptic peptides; P-VII has an additional methionine residue which is not found in the major core protein. We propose that at least two of the adenovirus 2 virion components are derived by the cleavage of higher molecular weight precursor polypeptides.

Formation of a vaccinia virus structural polypeptide from a higher molecular weight precursor: inhibition by rifampicin.

A vaccinia virus core polypeptide, with a molecular weight of 76,000 and a relative deficiency in tryptophan, was shown by pulse-chase experiments to form from a precursor. The latter may be a rapidly labeled, 125,000-molecular weight, tryptophan-deficient, virus-induced polypeptide, which diminished in quantity during the chase period and was barely detectable after two to three hours. Rifampicin completely prevented the formation of the core polypeptide without inhibiting the synthesis of the precursor. A rifampicin-resistant vaccinia mutant was used to demonstrate the specificity of this effect. The sequence of events after the removal of the drug suggested that cleavage of the precursor occurs during the formation of the virus core. Rifampicin appears to act by interrupting earlier maturational events which precede the formation of the core polypeptide.