Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Ficellomycin and feldamycin; inhibitors of bacterial semiconservative DNA replication.

The two peptide-like antibiotics ficellomycin and feldamycin impair semiconservative DNA replication but not DNA repair synthesis in bacteria. Specifically both antibiotics cause the accumulation of a 34S DNA species in toluenized Escherichia coli cells which lacks the capability of being integrated into larger DNA pieces and eventually the complete bacterial chromosome. Novobiocin, a known inhibitor of replicative DNA synthesis, was investigated for comparative purposes. The action of this latter antibiotic differs from the ones exerted by ficellomycin and feldamycin in the novobiocin appears to block an event associated with the initiation of Okazaki fragments. The fact that novobiocin impairs DNA gyrase suggests that this enzyme plays an essential role during the initiation of Okazaki pieces.     

Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.     

Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin.

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.     

Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.     

Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis.     

新生霉素抑制拓扑异构酶Ⅱ对多瘤病毒DNA合成的影响

本文报告了Ⅱ型拓扑异构酶抑制剂——新生霉素对多瘤病毒DNA合成的抑制作用,发现新生霉素对细胞和病毒DNA的体外合成具有不同的抑制曲线。新生霉素在复制过程中抑制复制中间物转变成成熟的病毒DNA分子的过程,同时影响病毒DNA分子的负超螺旋密度。本文结果提示Ⅱ型拓扑异构酶是病毒DNA复制末期所必须的酶。     

DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication.

Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.     

A cis-acting DNA signal for encapsidation of simian virus 40.

Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the sesdeleted plasmid 2 kbp away from the ori, suggesting that for encapsidation the two DNA elements have to be close to each other. These findings afford novel strategies for the investigation of viral encapsidation.     

C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich (167)RRRSQSPRR(175) Domain Is Critical for HBV Replication

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.     

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase alpha-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein-protein contacts during the entire initiation process.     

DNA synthesis in polyoma virus infection. III. Mechanism of inhibition of viral DNA replication by cycloheximide.

The formation of viral DNA was inhibited in polyoma virus-infected cells in which protein synthesis had been blocked by cycloheximide. The present studies show the following. (i) The pool of replicating viral DNA molecules was reduced in cycloheximide-treated cells by an amount consistent with inhibition of [3-H]thymidine incorporation into viral DNA, whereas the rate of turnover of the replicating population was not affected. (ii) The rate of conversion of replicating molecules into closed-circular DNA was not affected by cycloheximide. (iii) The rate of elongation of nascent viral DNA fragments into strands of unit genome length was unaffected by cycloheximide. It is concluded that viral DNA synthesis is inhibited in the absence of protein synthesis exclusively at the level of initiation of new rounds of genome replication. Replicating molecules already initiated at the time of addition of cycloheximide matured into progeny closed-circular DNA at a normal rate.     

Effect of aphidicolin on avian sarcoma virus replication.

We studied the effect of aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, on viral DNA replication and integration during the first 24 h after infection of quail embryo fibroblasts with avian sarcoma virus. In drug-treated cells, the synthesis of unintegrated linear viral DNA species was not impaired; however, the subsequent accumulation of circular viral DNA species and integrated proviral DNA was reversibly inhibited. After removal of the drug, circular viral DNA species were derived from preexisting linear viral DNA species, instead of being derived by de novo synthesis.     

Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.