Virazole prevents production of sindbis virus and virus-induced cytopathic effect in Aedes albopictus cells

The nucleoside analogue virazole (1-β-d-ribofuranosyl 1,2,4-triazole-3-carboxamide) at a concentration of 150 μg/ml inhibited the production of Sindbis virus (SV) and prevented virus-induced cytopathic effect (CPE) in Aedes albopictus LT C-7 cells. The drug had no effect on net viral RNA synthesis, the production of infectious RNA, or on nucleocapsid assembly. All viral structural proteins (E₁, E₂, and C) were synthesized and glycosylated in apparently normal amounts in the presence of virazole (Vz). However, most cells contained little or no reactive viral antigens as measured by an indirect immunofluorescent test. In contrast to the minimal effect on the synthesis of viral RNA and proteins, Vz markedly inhibited host macromolecular synthesis, as well as the growth of mosquito cells. In BHK21 cells the situation was different. As in C-7 cells, the drug caused a marked reduction in host macromolecular synthesis. However, CPE was not prevented and virus production was not inhibited. It is suggested that certain host functions may be required in the late stages of viral maturation and for expression of CPE and that these host functions are inhibited by Vz.     

DNA replication and late protein synthesis during SP82 infection of Bacillus subtilis

During infection of Bacillus subtilis by bacteriophage SP82, substantial quantities of at least two different late proteins were synthesized, even in the absence of detectable phage DNA replication. The two proteins were the lytic enzyme (which is possibly a host specific enzyme) and the protein(s) responsible for serum blocking power (which is presumably phage specific). The absence of replication was assured by the use of an SP82 double mutant carrying a mutation in each of two cistrons required for replication, and was verified by density transfer and isotope incorporation experiments. Thus, it appears that phage DNA replication is not required for late protein synthesis during SP82 infection.     

In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells.

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [³⁵S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.     

Membrane-associated proteins of T4-infected Escherichia coli

During the prereplicative period after the infection of Escherichia coli by phage T4, more than 50 proteins are synthesized. Many of them have been identified with their corresponding genes. Among them, at least 13 are selectively enriched in the membrane preparation. They include the products of the two rII genes, genes 39, 52 (DNA-delay), and others not yet identified. The majority of these proteins (90%) are extractable by the detergent, sarkosyl, and are possibly associated with the inner or cytoplasmic membrane. Based on their electrophoretic migration in SDS-polyacrylamide gels and on their tryptic fingerprints, these proteins are found to be phage induced. Two of the newly synthesized proteins that are selectively enriched in the cell wall or outer envelope fraction are found to be identical with two envelope proteins of the host cell. They are continually synthesized after phage infection although general host protein synthesis is shut off.     

A genetic analysis of bacteriophage λ head assembly

The sequence of steps involved in bacteriophage λ head assembly has been studied by characterizing second-site mutatons that can compensate for the reduced level of synthesis of a particular phage “head” protein. Such studies reveal these classes of protein interactions: (1) A reduced level of either pA synthesis or activity can compensate for the reduced amount of pD made when λDam mutants are grown in a supC host. This result implies that pA acts before pD during the packaging of phage DNA. (2) A reduced level of either pB synthesis or activity can compensate for the reduced amount of pE made when λEam mutants are grown in a supC host, and vice versa. This result suggests an interaction between these proteins that is stringently dependent on maintaining a proper ratio between them. An identical argument has been made to explain the properties of groE hosts (Sternberg, 1973). (3) A very specific class of mutations in phage gene E can compensate for the reduced amount of pC made when λCam mutants are grown in a supC host. This suggests that the products of these two genes directly interact.Using the techniques described in this report it is possible to easily isolate a variety of mutations (am, oc, ts, and missense) in specific phage “head” genes (genes A, B and E).     

The role of gene V protein in f1 single-strand synthesis

We demonstrate that, in cells infected with phage f1, only the gene V protein, a DNA-binding protein, is necessary to effect the switch from double- to single-strand synthesis; we specifically show that no host proteins are required for single-strand synthesis beyond those that are already required for double-strand synthesis. f1 temperature-sensitive (ts) gene II-infected cells were allowed to accumulate excess gene V protein in the absence of DNA replication through incubation at restrictive temperature. The infected cells were then shifted to permissive temperature in the presence of chloramphenicol; single-strand synthesis ensued. When, however, the identical experiment was performed with wild-type f1-infected cells, double, rather than single strands were synthesized. Since the principal difference between the two experiments lay in the accumulation of excess gene V protein in the tsII infection, single-strand synthesis must have halted in the wild-type infection because no free gene V protein was available and not because a chloramphenicol-sensitive host protein was being depleted. Chloramphenicol, by preventing protein synthesis, apparently blocks the two sources of gene V protein normally used for single-strand synthesis. No new gene V protein can be synthesized, and all previously synthesized gene V protein remains stably complexed with preexisting single strands rather than being recycled during morphogenesis of the single strands. In the absence of both new and recycled gene V protein, double-strand synthesis resumes.If a failure to recycle gene V protein were indeed the lesion induced by chloramphenicol treatment, then inactivation of specific f1 gene products required for morphogenesis, through use of amber and temperature-sensitive mutant phage, might also lead to replacement of single-strand synthesis by double-strand synthesis. Such cessation of single-strand synthesis was indeed found after infections by mutants in genes I, IV, VII, and VIII but not after infections by mutants in genes III and VI. The gene III and VI proteins therefore act at a later step in morphogenesis than the gene I, IV, VII, and VIII proteins; they appear to function after the single strands and gene V protein have separated. Data obtained from temperature shift experiments indicate that the supply of gene V protein in an infected cell is carefully regulated and that there is never much free gene V protein available.     

Analysis of Yaba tumor poxvirus-induced proteins in infected cells by two-dimensional gel electrophoresis

Lack of inhibition of host protein synthesis by Yaba tumor poxvirus and comigration of viral and host proteins did not interfere with high resolution of virus-induced proteins when two-dimensional gel electrophoresis was employed. A total of 84 virus-induced proteins, 44 structural and 40 nonstructural, comprising 62.9% of the coding capacity of Yaba virus DNA, were detected. These proteins may be further categorized in two classes: early, synthesized in the presence of DNA inhibitor, Ara-c; and late, beginning at 6 hr p.i. with the bulk of viral protein synthesis occurring later than 30 hr p.i. The rate of synthesis of viral proteins fell into two patterns: group A, in which the rate of synthesis decreased; and, group B, in which the rate of synthesis increased after the initial appearance of the protein.     

DNA replication in bacteriophage φ29: The requirement of a viral-specific product for association of φ29 DNA with the cell membrane of Bacillus amyloliquefaciens

A rapidly sedimenting complex has been isolated from sheared lysates of φ29-infected Bacillus amyloliquefaciens by sedimentation on linear density gradients of Renografin (0–38%) containing an underlayer of 76% Renografin. The complex, previously identified as membrane-bound bacterial DNA, contains parentally labeled viral DNA and is enriched for pulse-labeled viral DNA. DNA synthesized early in infection is a precursor to DNA found free in the cytoplasm and in mature phage particles. The association of parental phage DNA with the complex is detectable near the onset of viral DNA replication. The maximum amount of parental φ29 DNA associating with the cell membrane occurs by the middle of the DNA synthesis period. This association can be inhibited by actinomycin D or rifamycin if added 5 min but not if added 10 min after infection. Chloramphenicol or puromycin added 10 min before virus, reduces the amount of association of parental DNA with the complex fourfold. A mutant of φ29 is unable to replicate its DNA at 43 C and cannot associate with the complex at this temperature unless co-infected with wild-type phage. Since formation of the complex only occurs when viral DNA is being synthesized, the complex may be a necessary intermediate in the replication of φ29 DNA. The viral mutation TS35 appears to affect a protein whose function is to associate infecting viral DNA with the host cell membrane.     

Nonsense mutants of the lipid-containing bacteriophage PR4

Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated. One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles. We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells. Finally each complementation group was tested for the ability to lyse an Su- host. We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme.     

Involvement of the htpR gene product of Escherichia coli in phage λ development

Growth of phage λ at high temperature requires a functional htpR host gene. The stages of the phage growth cycle shown to be dependent on htpR gene function include prophage excision and particle morphogenesis. Two types of morphogenetic abnormalities have been detected. One is a defect in phage tail assembly that results from a deficiency in tail fibers even though gpJ is produced. The severity of this defect is phage-strain specific. The second morphogenetic defect is less clearly defined, but results in formation of aberrant phage head structures. These abnormalities in λ reproduction are presumed to be caused by the absence in htpR mutant host cells at high temperature of one or more of the heat-shock proteins of Escherichia coli whose synthesis is known to be regulated by the htpR gene.     

Synthesis of VSV RNPs in vitro by cellular VSV RNPs added to uninfected HeLa cell extracts: VSV protein requirements for replication in vitro

Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited.     

f1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

The phospholipid composition of cytoplasmic membranes prepared from bacteria grown in the presence of [³²P]phosphate and infected with f1 wild type and f1 amber mutant bacteriophages was determined. Ninety minutes after infection with f1 amber mutants in genes 1, 3, 4, 5, 6, and 7 the percentage of cardiolipin was increased from the level in uninfected bacteria of 5% to about 20–35%, and the percentage of phosphatidylethanolamine was decreased from 70% to about 50–60%. The phospholipid composition of cytoplasmic membranes from bacteria infected with a phage containing an amber mutation in the coat cistron (gene 8) did not differ from that of uninfected bacteria. Although late in infection there were no detectable alterations in phospholipid metabolism in wild type infected bacteria, transient alterations in phospholipid metabolism occurred in these bacteria 10 to 20 min after infection. During this time period, the f1 coat protein was found to be rapidly synthesized but was not being packaged into mature phage and released from bacteria. Both the long-term alterations of phospholipid metabolism found in the amber mutant infected bacteria and the transient alterations found in wild-type infected bacteria resulted from an increase in the rate of synthesis of phosphatidylglycerol and cardiolipin and a decrease in the rate of synthesis of phosphatidylethanolamine. These results are discussed in terms of the relationship between the accumulation of f1 coat protein in infected bacteria and the observed alterations in phospholipid metabolism.     

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

A sedimentation analysis of DNA found in Escherichia coli infected with phage lambda mutants

A study was made of the phage DNA produced in Escherichia coli infected with mutants of λ unable to produce infectious particles because of defects in genes controlling head formation. Bacteria were irradiated with UV to eliminate host DNA synthesis and then infected with various λ sus mutants under conditions where subsequent radioactive labelling appeared exclusively in phage DNA. The intracellular DNA was extracted and examined by sedimentation in neutral and alkaline sucrose gradients. Mutants in genes A, B, C, D, and E cause blocks in DNA maturation which result in the accumulation of “rapidly sedimenting” species. Mutants in genes W and F accumulate much less of the rapidly sedimenting DNA and synthesize about 50–100% of the normal amounts of mature molecules as found in control infections. Intracellular phage DNA was purified from host material by density labelling and examined by electron microscopy. The “rapidly sedimenting” species appeared to be linear concatamers with lengths up to and exceeding four units. In an A mutant circular molecules were also found; these were both of monomer and multimer length (dimers and trimers). The proportion of immature DNA found by electron microscopy (long molecules) and by sedimentation (fast sedimenting DNA) agreed very well.     

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA^? mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3⁺) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642.At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion (R. Goorha and A. Granoff, 1974, Virology60, 237–250) that in FV3⁺-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.