Studies on the polypeptides of poxvirus. I. Comparison of structural polypeptides in vaccina, cowpox and Shope fibroma viruses.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.     

Shope Fibroma Virus I. Biological and Molecular Properties of a Cytocidal and a Noncytocidal Strain

The biological and molecular properties of two strains of Shope fibroma virus (SFV) were compared. SFV-I was highly cytocidal to most of the cell lines tested and produced pocks in the chorioallantoic membrane of chick embryos. By contrast, SFV-W did not produce cytopathic effects in any of the cell lines or in the chorioallantoic membrane, but it induced characteristic foci 3 to 4 days after infection. Both strains produced tumors when inoculated into the skin of susceptible rabbits. Maximal infectivity in BSC-1 cells was reached by both strains between 24 to 48 h after inoculation. Viral DNA synthesis also took place at the same time, although cells infected with SFV-I incorporated three times more [(3)H]thymidine than cells infected with SFV-W. Sedimentation analysis and hydroxylapatite chromatography of the two viral DNAs indicated that their molecular weights were similar and that both were naturally cross-linked. Digestion with three restriction endonucleases, however, revealed that they had different restriction sites. When SFV-I and vaccinia DNA were compared, the restriction patterns were more alike. Analysis of the virion structural proteins by gel electrophoresis indicated that SFV-I, SFV-W, and vaccinia virus had many polypeptides in common, although there were distinctive differences among the three viruses. Finally, the results of plaque neutralization tests with different antisera showed that SFV-I and SFV-W shared common antigens and that vaccinia antiserum inhibited SFV-I but not SFV-W. We conclude that the SFV-I genome contains information for both cytolysis and tumorigenesis. This unusual virus may be a recombinant between an orthopoxvirus and a leporipoxvirus.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.     

Deletion of a 9,000-base-pair segment of the vaccinia virus genome that encodes nonessential polypeptides.

Deletions contained within the genomes of unstable and stable variants of vaccinia virus (strain WR) were analyzed. Restriction endonuclease mapping and hybridization to specific 32P-labeled DNA probes indicated that more than 6 X 10(6) daltons of DNA were deleted from the variants. In each case, the deletion occurred on the left side of the genome and started very close to the junction of the inverted terminal repetition and unique sequence. Both variants also contained a new SstI side on the right side of the genome. Hybridization selection and cell-free translation experiments indicated that these variants lost the ability to synthesize at least eight early mRNA's mapping within the deleted region. Although the deleted DNA was not essential for replication of the WR strain of vaccinia virus under laboratory conditions of infection, it presumably has a defined role under other circumstances. This conclusion was based on the conservation within the Elstree strain of vaccinia, the Utrecht strain of rabbitpox, and the Brighton strain of cowpox virus of sequences homologous to the deleted DNA. Moreover, mRNA's that hybridized to the deleted vaccinia virus DNA segment and encoded similar size polypeptides were made in cells infected with rabbitpox and cowpox viruses.     

The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome.

We have mapped early and late viral gene products expressed in Autographa californica nuclear polyhedrosis virus ( AcNPV )-infected Spodoptera frugiperda cells by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of AcNPV DNA. Proteins synthesized in vitro were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. At least four early AcNPV -specific polypeptides were found which mapped in two regions of the genome (9-25 and 43-59 map units). These early mRNAs are also synthesized at late times in the infection cycle. Cell-free translation of restriction fragment-selected late AcNPV -specific RNA (24 h post-infection) resulted in the identification and mapping of 24 viral proteins. Curiously, the region between approximately 70 and 80 map units on the viral genome has been found silent with respect to mRNA which is translatable in a cell-free system. However, there may be RNA transcribed from this viral DNA segment.     

Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis.

The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various times postinfection were also analyzed. At least 13 proteins labeled with [3H]glucosamine were detected in vaccinia-infected HeLa cells.     

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.     

Studies on the polypeptides of varicella-zoster (V-Z) virus. 1. Detection of varicella-zoster virus polypeptides in infected cells.

Virus-induced polypeptides in cells infected with varicella-zoster virus (VZV) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. When human embryonic lung (HEL) cells infected with the Oka strain of VZV were labelled with 35S-methionine or 14C-glucosamine from 40 hr to 46 hr after infection, at least 18 VZV-induced polypeptides and 10 glycoproteins could be identified in the infected cells. The molecular weights of the polypeptides and glycoproteins ranged from about 145,000 to 23,000, and from about 105,000 to 48,000, respectively. Lysates of VZV-infected cells were treated with specific antisera prepared in green monkeys or guinea-pigs, and analysed by SDS-PAGE and fluorography. In all, 33 polypeptides (with molecular weight of about 145,000 to 22,000) and 13 glycoproteins (molecular weight, about 105,000 to 38,000) were found in the immunoprecipitates. None of these polypeptides and glycoproteins were detected when infected cells cultured in the presence of phosphonoacetic acid (PAA) were treated in the same way.     

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.     

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Polyamines in vaccinia virions and polypeptides released from viral cores by acid extraction.

Vaccinia virions propagated in the presence of [3H]ornithine were found to contain two labeled polyamines, spermine and spermidine. In complete virions the ratio of radioactively labeled spermine to spermidine was about 1:10, whereas in viral cores the ratio was 2:5. This suggests that some spermidine was preferentially lost during the conversion of virions to cores or that spermidine was present in the virions both inside and outside the core structure. Addition of [3H]ornithine to vaccinia virus-infected cells as late as 6 h postinfection demonstrated that, although the conversion of this precursor to polyamines was reduced by 50% or more as compared to mock-infected cells, complete inhibition of polyamine synthesis did not occur. Two percent or less of the total radioactivity associated with virions grown in the presence of [3H]ornithine was found to be acid soluble. Polyacrylamide gel electrophoretic analysis showed that all the structural polypeptides were labeled when virions were propagated in the presence of [3H]ornithine. When cores labeled with a mixture of 14C-labeled amino acids were extracted with 0.25 N H2SO4, 12 to 15% of the labeled core polypeptides were released and could be precipitated with acetone. About 40% of [3H]arginine-labeled polypeptides associated with cores were extracted with acid. Four polypeptides or groups of polypeptides were resolved after polyacrylamide gel electrophoresis analysis of the acid-soluble fraction of cores with molecular weights of about 58,000, 34,000, 24,000 and 10,000 to 12,000. About 40% of the [3H]arginine radioactivity extracted from cores coelectrophoresed with the 10,000 to 12,000-molecular weight polypeptide, indicating that this may represent an arginine-rich, histone-like structural polypeptide of the virion.     

The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha.

Degenerate oligonucleotide probes corresponding to a highly conserved region common to epidermal growth factor, transforming growth factor alpha, and vaccinia growth factor were used to identify a novel growth factor gene in the Shope fibroma virus genome. Sequence analysis indicates that the Shope fibroma growth factor is a distinct new member of this family of growth factors.     

Turnover of cytokeratin polypeptides in mouse hepatocytes

The turnover of cytokeratin polypeptides A (equivalent to No. 8 of the human cytokeratin catalog) and D (equivalent to human cytokeratin No. 18) of mouse hepatocytes was studied by pulse-labeling of mouse liver proteins after intraperitoneal injection of L-[guanido-14C]arginine and [14C]sodium bicarbonate. At various times after injection cytoskeletal proteins were prepared and separated by SDS-polyacrylamide gel electrophoresis, and the specific radioactivities of polypeptides recovered from excised gel slices were determined. With L-[guanido-14C]arginine a rapid increase in the specific radioactivity of both cytokeratins was observed which reached a plateau between 12 and 24 h. With [14C]sodium bicarbonate maximal specific radioactivity was obtained at 6 h followed by a rapid decrease to half maximum values within the subsequent 6 h and then a slower decrease. Half-lives were determined from the decrease of specific radioactivities after pulse-labeling by least-squares plots and found to be 84 h (for cytokeratin component A) and 104 h (component D) for arginine labeling. Values obtained after bicarbonate labeling were similar (95 h for A and 98 h for D). These results show that liver cytokeratins are relatively stable proteins and suggest that components A and D are synthesized and degraded at similar rates, probably in a coordinate way.     

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with ³⁵S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with ¹⁴C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.     

Structural polypeptides of rabbit, bovine, and human papillomaviruses.

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.     

Differential synthesis of polypeptides during morphogenesis of Mucor.

The extent of differential gene expression during morphogenesis of Mucor racemosus was investigated by two-dimensional polyacrylamide gel electrophoresis of neutral and acidic polypeptides. Cellular proteins were labeled with [35S]methionine in cells growing in either the yeast or hyphal form, or in yeast cells undergoing the transition of hyphae. The results showed that of the 400 to 500 polypeptides resolved by electrophoresis, relatively few were specific to one or the other morphological form. The major change in the patterns of proteins synthesized during morphogenesis was a change in rates of synthesis of individual polypeptides. Experiments in which morphogenesis was affected under aerobic or anaerobic conditions showed that the majority of changes in the protein patterns were associated with morphogenesis and were not a specific response to O2.     

Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA.

Summary Using purified yeast mitochondrial DNA as a template forE. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low backgroundE. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochromec oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26×10³ daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.