Inhibition Of Host DNA Synthesis Research Articles

Human Cytomegalovirus Protein pUL117 Targets the Mini-Chromosome Maintenance Complex and Suppresses Cellular DNA Synthesis

Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.

Nuclear targeting of Semliki Forest virus nsP2.

The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.

Physical mapping of two herpes simplex virus type 1 host shutoff loci: rescue of each ts mutation occurs with two unique cloned regions of the viral genome.

Two complementing temperature-sensitive (ts) herpes simplex virus type 1 (HSV-1) mutants, PAA1rts1 and ts199, were defective in viral DNA synthesis and in the shutoff of cellular macromolecular synthesis at 39.5 degrees C, the nonpermissive temperature. PAA1sts1 and PAA1rts1+ recombinants and PAA1rts1+ revertants were used to examine the contributions of the PAA1r mutation and the ts1 mutation of PAA1rts1 in affecting the levels of viral and cellular DNA synthesized at 34 and 39.5 degrees C. The results of this study suggests an interaction between the viral DNA polymerase and the ts1+ gene product during HSV-1 DNA replication and possibly in the inhibition of host DNA synthesis by HSV-1. Physical mapping of the ts mutations present in ts199 and the PAA1sts1 recombinant ts1-8 were performed by intratypic marker rescue experiments. Surprisingly, both the ts1-8 and ts199 mutations were rescued by two cloned fragments: ts1-8 by BglII-K (map coordinates 0.095 to 0.163) and BglII-I (map coordinates 0.314 to 0.417), while ts199 was rescued by BglII-K and BglII-O (map coordinates 0.163 to 0.197). In more refined mapping experiments, the regions between coordinates 0.347 to 0.378 and 0.126 to 0.163 were able to rescue the ts1-8 mutation. Southern hybridization analysis confirmed that the fragments that rescued ts1-8 and those that rescued ts199 had homology, as predicted by the physical mapping results.

The morphology of staphylococcal bacteriophage K and DNA metabolism in infected Staphylococcus aureus.

The morphology and dimensions of bacteriophage K particles were determined by electron microscopy. This virus had an icosahedral head (approx. 70 nm diam.) and a long (210 nm) thin (15 nm) contractile tail which terminated in a complex basal appendage. The precise dimensions of the particles were dependent on the negative stain employed. The buoyant densities of the K virus particle and its DNA were 1 X 479 g/ml and 1 X 689 g/ml respectively. The DNA had a base composition of 30% G + C, a contour length of 16 X 1 micron and a calculated mol. wt. of 33 X 10(6). With Staphylococcus aureus (NCTC 9318) as host, the latent period was 25 min, the eclipse period 14 min and the average burst size 60 p.f.u./bacterium. Infection resulted in inhibition of host DNA synthesis and degradation of the bacterial DNA: the products were used for tee synthesis of phage DNA. The kinetics of DNA synthesis is infected and uninfected bacteria were examined. There was no initial cessation of DNA synthesis in the infected bacteria.

Inhibition of host DNA synthesis by adenovirus infection is reversed at elevated temperatures.

Infection of KB cells by adenovirus normally results in a drastic reduction of host DNA synthesis at 33 to 37 degrees C. This inhibition was largely reversed by raising the temperature to 41 degrees C. Inhibition was reinstated if the temperature is lowered.

Carcinogens enhance survival of UV-irradiated simian virus 40 in treated monkey kidney cells: induction of a recovery pathway?

Treatment of monkey kidney cells with low doses of carcinogen enhances the survival of UV-irradiated simian virus 40 (SV40). This is true for compounds with UV-like effects (metabolites of aflatoxin B1, N-acetoxyacetylaminofluorene) and compounds with x-ray-like effects (methyl methanesulfonate, ethyl methanesulfonate). This phenomenon resembles the UV-reactivation of viruses in eukaryotic cells. The carcinogen-induced enhancement of the survival of UV-irradiated SV40 is correlated with the inhibition of host-cell DNA synthesis, suggesting that the inhibition is an inducing agent. An enhancement of UV-irradiated SV40 survival is also obtained in cells treated with hydroxyurea or cycloheximide for long enough that there is still inhibition of host DNA synthesis during the early stage of SV40 infection. We hypothesize that treatment of host cells with carcinogens induces a new recovery pathway that facilitates the replication of damaged DNA, bypassing the lesions and resulting in the enhanced survival of UV-irradiated SV40. This inducible process might represent the expression of "SOS repair" functions in eukaryotic cells analogous to the previously demonstrated induction of SOS repair in bacteria after UV or carcinogen treatment.

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.

Cold-sensitive Pseudomonas RNA polymerase. I. Characterization of the host dependent cold-sensitive restriction of phage CB3.

Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells.

Outgrowth of Bacillus cereus spores harboring bacteriophage CP-51 DNA. I. Initiation of bacteriophage development.

Time of initiation of bacteriophage CP-51 RNA synthesis, inhibition of host DNA synthesis, initiation of CP-51 DNA synthesis, and release of phage was determined in infected Bacillus cereus cells and outgrowing spores harboring phage DNA. The development of phage in outgrowing spores is initiated 45 min after induction of spore germination and then proceeds at a rate similar to that observed in infected cells. Although spore RNA synthesis is inhibited by the presence of chloramphenicol or puromycin during germination, these antibiotics do not inhibit initiation of phage DNA transcription in outgrowing spores.

The kinetics of bacterial DNA synthesis during indirect induction of prophage lambda.

The possibility that the processes of direct and indirect induction of prophage λ can be unified on the basis of a temporary inhibition of bacterial DNA synthesis has been examined. The kinetics of DNA synthesis in non-lysogenic bacteria were not significantly affected during a 60 min mating with UV-irradiated ColI drd donor cells. In contrast, direct irradiation of the bacteria with a UV dose which induced phage development with an efficiency similar to that obtained by indirect induction caused an abrupt cessation of DNA synthesis lasting for about 12 min. It is concluded that indirect induction of prophage λ differs from most treatments which induce directly in that it is not associated with an inhibition of host DNA synthesis.

Control of DNA synthesis in Bacillus subtilis by phage ∅e

After infection of Bacillus subtilis 3610 with bacteriophage ∅e, which contains hydroxymethyluracil in place of thymine in its DNA, bacterial DNA synthesis is arrested. Our results show that the phage-coded deoxythymidine triphosphate hydrolase (TTPase) is not essential for the arrest. A phage-coded protein made in the first 13 min after infection appears to be involved in arresting host DNA synthesis. Phage mutants defective in this function have been isolated. The inhibition of host DNA synthesis does not appear to take place by detachment of bacterial DNA from the cell membrane or by inhibition of DNA polymerase. A phage фe mutant defective in TTPase, with thymine replacing up to 20% of the HMU in its DNA, can grow and transfer the thymine-containing DNA to progeny normally. The role of HMU in this phage remains unexplained.

Inhibition of bacterial DNA and protein synthesis in Bacillus subtilis by phage SP82. Effect of changes of temperature on the inhibition

Data are presented concerning the effect of raising the temperature from 37° to 45° on the inhibition by phage SP82 of protein and DNA synthesis in Bacillus subtilis SH. When the bacteria are incubated for 40 min at 45° before infection, infection proceeds as at 37°: host protein and DNA synthesis are inhibited. When the bacteria are incubated for less than 30 min at 45° before infection, synthesis of some host proteins is not inhibited and inhibition of host DNA synthesis is delayed. The data presented suggest that the inhibition of host DNA synthesis may result directly from the reduction of thymidine triphosphate pool size after infection. Temperature jump experiments suggest some general requirements which would have to be satisfied by any model of inhibition of host protein synthesis in this system.

Synthesis of DNA in phage-infected Bacillus subtilis

The hydroxymethyl-uracil-containing bacteriophage ∅e causes inhibition of host DNA synthesis in Bacillus subtilis 3610. This inhibition is not accompanied by breakdown of the host DNA to acid-soluble form nor by production of double-strand breaks in it. The inhibition of thymidylate synthetase produced by this phage is not responsible for the arrest of host DNA synthesis observed after 6 minutes of infection at 37° since the arrest is still observed in bacterial mutants requiring and actively using exogenous thymine. The role of the phage-induced deoxythymidine triphosphate nucleotidohydrolase (dTTPase) in producing the arrest of host DNA synthesis has been investigated using phage mutants which induce temperature-sensitive dTTPase. Even under conditions in which the dTTPase level is low enough to permit incorporation of some thymine into the DNA of the phage, there is still an inhibition of host-DNA synthesis at the usual time after infection. The results suggest, but do not prove, that the phage may give rise to some additional mechanism, besides the induction of dTTPase, resulting in inhibition of host DNA. It is not yet known whether the phage particles with thymine-containing DNA are normally functional.