Inhibition of adenovirus replication by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

The halogenated benzimidazole derivative 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) inhibits reversibly the replication of human adenovirus type 2 (Ad2) and its DNA in human KB cells. Viral DNA replication is almost completely blocked when the drug is added earlier than 4 hr postinfection in concentrations between 50 and 150 μM. Replication of viral DNA in all four size-classes (>100 S, 50–90 S, 34 S, and <20 S) is inhibited. The replication block is reversible upon withdrawal of the inhibitor. When DRB is administered at the time of maximal viral DNA replication, 16–18 hr postinfection, the inhibitor has no apparent effect on viral DNA synthesis. In the presence of 150 μM DRB, synthesis of early virus-specific RNA in the nucleus is reduced by approximately 90% and the appearance of virus-specific RNA sequences in the cytoplasm is reduced by >95%, as demonstrated by DNA-RNA filter hybridization. Thus, the block in viral DNA replication is best explained by the inhibition of the synthesis of early virus-specific RNA.     

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.     

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.     

Bleomycin, an inhibitor of vaccinia virus replication

Bleomycin inhibits vaccinia virus replication in infected HeLa cells and in mice. Viral DNA synthesis and core RNA polymerase activity are significantly inhibited by the antibiotic. Synthesis of messenger RNA in isolated vaccinia cores is inhibited, but synthesis of poly(A) is unaffected. Vaccinia core DNA is degraded during the course of the RNA polymerase reaction in the presence of bleomycin. The presence of ribonucleoside triphosphates accelerates the degradation of vaccinia core DNA by bleomycin. Effects on DNA synthesis, in particular, the induced degradation of template DNA, may be responsible for the observed antiviral activity of bleomycin.     

Effect of lysosomotropic compounds on early events in foot-and-mouth disease virus replication

The effect of three lysosomotropic compounds, chloroquine, monensin and NH4Cl, on the replication of foot-and-mouth disease virus (FMDV) type A12 was studied. Viral replication was almost totally inhibited by 0.5 mM chloroquine, 50 microM monensin, or 25 mM NH4Cl. Monensin and NH4Cl affected replication when added either before or within the first hour of infection. Chloroquine, however, still inhibited viral replication when added up to 2.5 h after infection. Assays of binding of radiolabeled virus to cells showed that these compounds had no effect on viral adsorption. Neither monensin nor NH4Cl had any significant effect on cellular protein synthesis, but there was no evidence of viral protein synthesis in cells infected in the presence of these compounds. In contrast, chloroquine inhibited both cellular and viral protein synthesis. Eclipse assays, performed in the presence of the compounds, showed that while chloroquine and NH4Cl had little effect on cell-induced degradation of incoming virions to 12 S protein subunits, monensin inhibited this reaction. The replication of representative members of all seven serotypes of FMDV was inhibited by monensin although some types were less sensitive to the compound than others. These results are consistent with a model which postulates that viral eclipse is the result of acidification of endocytic vesicles which degrade entrapped virions to 12 S protein subunits resulting in the release of genome RNA.     

Azathioprine inhibits vaccinia virus replication in both BSC-40 and RAG cell lines acting on different stages of virus cycle

In the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage.     

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.     

Biosynthesis and post-translational cleavage of vaccinia virus structural protein VP8

We have obtained antiserum against highly purified vaccinia virus structural protein VP8, a major DNA binding protein present in the viral core particle. The antiserum has been used to monitor the course of the biosynthesis of this protein. The protein can first be detected in extracts of infected cells at 4 hr postinfection (p.i.). Its synthesis increases significantly at 5 hr p.i. and is maintained at about the same level up to 11 hr. The requirement of viral DNA replication for VP8 synthesis indicates that it is a viral late protein. This protein is synthesized in the form of a 28-kDa precursor, which is then processed to a 25-kDa product. The half-life of the precursor is about 2 hr. Comparing the N-terminal amino acid sequence of this purified protein with those derived from the published DNA sequence of the vaccinia viral genome [J.P. Weir and B. Moss (1984) J. Virol. 51, 662-669], it is found that VP8 maps to the HindIII L fragment of the viral genome. The cleavage site at which processing takes place lies between amino acids 32 (Gly) and 33 (Ala) from the N-terminal end of the precursor.     

The effect of lithium chloride on the replication ofHerpes simplex virus

Lithium chloride inhibited the replication of type 1 and type 2Herpes simplex virus at concentrations which permitted host cell replication. Virus polypeptide and antigen synthesis were unaffected while viral DNA synthesis was inhibited. The replication of two other DNA viruses, pseudorabies and vaccinia virus, was inhibited but there was no inhibition of two RNA viruses, namely, EMC and influenza virus.     

Interferon-gamma-induced assembly block in the replication cycle of adenovirus 2: augmentation by tumour necrosis factor-alpha.

Replication of adenovirus 2 (Ad-2) is inhibited in A 549 cells pretreated with interferon-gamma (IFN-gamma). The antiviral effect is synergistically enhanced by the simultaneous presence of tumor necrosis factor-alpha (TNF-alpha) before infection. Under conditions of strong inhibition of virus progeny formation, viral DNA synthesis and [35S]methionine incorporation into most late viral proteins are only marginally impaired. Pulse chase experiments indicate a partial inhibition of processing of viral proteins. Viral proteins are not degraded and capsomeres accumulate in the inhibitor-treated cells. Capsid formation, on the other hand, is strongly inhibited in the cytokine-treated cells. The inhibition of Ad-2 replication in A 549 cells by IFN-gamma and TNF-alpha is caused, therefore, by a block in the maturation of Ad-2.     

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.     

Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus

Methods have been developed for rapid isolation and genetic analysis of vaccinia virus mutants. These methods include: (1) monitoring mutagenesis by measuring conversion of wild-type, phosphonoacetic acid-sensitive virus to phosphonoacetic acid-resistant virus, (2) screening for mutants by plaque enlargement, and (3) a qualitative spot test for complementation. Twenty-six temperature-sensitive mutants of vaccinia virus have been isolated. All have reversion indices of 10^?4 or less. One-step growth experiments have been done at 40° and 31° with all the mutants and in all cases the virus yield at 40° is less than 8% of the yield observed at 31°. Complementation analysis has been completed on all 26 mutants, showing that these mutants together comprise 16 complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize viral DNA at the nonpermissive temperature. The results show that 3 of the 24 have a DNA-negative phenotype. These three mutants fall into two complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize early and late viral proteins at the nonpermissive temperature. From this analysis, four phenotypes appear: (1) normal, (2) a phenotype associated with DNA-negative mutants characterized by prolonged synthesis of early proteins and the absence of late protein synthesis, (3) weak or slow late protein synthesis, (4) abortive late protein synthesis.     

Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice.

We have analyzed the role of exogenous administration of mouse interferon (IFN alpha + beta) on the replication of vaccinia virus in peritoneal cells and in the spleen of Balb/c mice. Mice were pretreated for 16 hr with IFN and then infected with a vaccinia virus recombinant expressing luciferase under an early or late virus promoter, and the enzyme activity was measured in the course of virus infection. A dose of IFN as low as 10(3) units/mouse abolished the appearance of luciferase activity in cells of the peritoneal cavity and in spleen cells. The IFN-mediated inhibition of luciferase activity was observed even when mice were infected 4 days after the administration of IFN. The IFN-treated animals were considered free of virus since neither luciferase nor viral proteins were detected in target cells several days after virus infection. Despite a severe IFN-mediated inhibition of luciferase activity, the appearance of luciferase on mRNA levels was not inhibited 6 hr after virus infection. Our finding revealed that replication of vaccinia virus in Balb/c mice is exquisitively sensitive to inhibition by IFN and that this effect occurs at early times postinfection, most likely as a result of a translational block.     

Inhibition by monensin of human cytomegalovirus DNA replication

Summary Monensin, at concentrations which depended on the multiplicity of infection, was found to prevent DNA replication of human cytomegalovirus (HCMV) as well as production of viral progeny in human foreskin fibroblasts. The drug did not affect DNA replication of herpes simplex virus. Inhibition of consecutive HCMV DNA synthesis was also observed following delayed addition of the drug within 12–24 hours postinfection, but was fully reversible upon its removal. Viral replication proceeded, however, without impairment in cultures treated with monensin prior to infection. Induction of viral DNA polymerase activity was not impeded by the inhibitor. Analysis of protein- and glycoprotein synthesis revealed that monensin interfered with the production of a number of HCMV-specific polypeptides. Furthermore, evidence was obtained that the drug may hinder intracellular transport of a 135 kd glycopolypeptide.With 6 Figures     

Biosynthesis and phosphorylation of vaccinia virus structural protein VP11

One of the major DNA binding proteins contained in vaccinia virus is an 11-kDA species denoted VP11. The biosynthesis of VP11, a late polypeptide, occurs subsequent to the initiation of viral DNA replication. In particular, VP11 synthesis is blocked by cytosine arabinoside, a specific inhibitor of DNA synthesis. We show here that VP11 is specifically phosphorylated subsequent to translation. Phosphorylated VP11 is present both in viral core particles and in the cytoplasm of virus-infected cells. Kinetic analysis reveals that the total amount of phosphorylated VP11 species increases rapidly and remains approximately constant for as long as 17 hr postinfection. Phosphorylation occurs at two different serine residues, progressing from either site singly to the diphosphorylated product. Under steady-state conditions, the phosphorylated derivative constitutes approximately 85% of total VP11 in extracts of vaccinia virus-infected cells. Even though 15% of the VP11 remains unphosphorylated in cell extracts, only phosphorylated VP11 is found in mature viral cores.     

Effect of interferon on the exogenous Friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65^gag, and glycoprotein precursor, gPr85^env, to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65^gag, was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.