Inhibition Of Viral DNA Replication Research Articles

Frog Virus 3 Replication: Induction and Intracellular Distribution of Polypeptides in Infected Cells

The synthesis of the polypeptides induced in frog virus 3-infected cells was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled cell extracts. Purified frog virus 3 contained 22 polypeptides, with molecular weights in the range 9 x 10(3) to 114 x 10(3). All of the structural and an additional seven nonstructural polypeptides were detected in infected cell lysates. The following three classes of induced polypeptides (under temporal control) were observed in BHK cells: at 2 h, four alpha polypeptides; at 4 h, 13 beta polypeptides; and at 6 h, the remaining 12 gamma polypeptides. The total molecular weight of the infected cell-specific polypeptides (ICPs) was approximately 1.5 x 10(6), which accounts for about 30% of the coding capacity of the viral genome. At least 10 of the induced polypeptides were phosphorylated, but none was glycosylated or sulfated. No evidence for posttranslation cleavage of polypeptides in pulse-chase and inhibition experiments was obtained. The synthesis of gamma polypeptides was not detected in the presence of the viral DNA replication inhibitors cytosine arabinoside and hydroxyurea, but halogenated nucleotides apparently had no effect. These results suggest that alpha and beta polypeptides are "early" events and that detectable gamma polypeptide synthesis is dependent on the production of progeny viral DNA. The regulation of frog virus 3-induced polypeptide synthesis in infected BHK cells was examined by using inhibitors of protein and RNA synthesis and amino acid analogs. These experiments confirmed the existence of three sequentially synthesized, coordinately regulated classes of polypeptides, designated alpha, beta, and gamma. The requirements for the synthesis of each class were as follows: (i) alpha polypeptides did not require previous cell protein synthesis; (ii) beta polypeptides required a prescribed period of alpha polypeptide synthesis and new mRNA synthesis; and (iii) gamma polypeptides required prior synthesis of functional beta polypeptides and new mRNA synthesis. alpha polypeptide synthesis was controlled by beta and gamma polypeptides, and alpha and beta polypeptides were involved in the suppression of host cell polypeptide synthesis. Indirect evidence was obtained for the temporal regulation of frog virus 3 transcription. The intracellular distribution of virus-induced polypeptides in cells infected with frog virus 3 was investigated by using standard cell fractionation techniques. Most of the 29 induced polypeptides were bound to structures within the nucleus, and only two ICPs were not associated with purified nuclei. When isolated nuclei were incubated in an infected cell cytoplasm preparation, all of the nuclear ICPs were incorporated in vitro. All of the ICPs were associated with ribosomal and rough endoplasmic reticulum fractions of infected cells, and a number of ICPs were found on smooth intracellular membranes. Most of the ICPs were also associated with purified plasma membranes of infected cells, and one polypeptide (ICP 58) was highly enriched in the plasma membrane compared with whole cell extracts or purified frog virus 3.

Synthesis of polynucleotide 5'-triphosphatase in vaccinia virus-infected HeLa cells.

Synthesis of polynucleotide 5'-triphosphatase, which is presumably involved in the initial modification in the series of reactions by which 5'-termini of vaccinia mRNA become capped and methylated, has been demonstrated in vaccinia virus infected HeLa cells. Synthesis of the enzyme is prevented by actinomycin D and cycloheximide, suggesting that both de novo DNA-dependent RNA and protein syntheses are required. On the other hand, cytosine arabinoside, an inhibitor of viral DNA replication, does not prevent induction of the enzyme. The latter observation, together with the kinetics of synthesis of the enzyme in vaccinia virus-infected HeLa cells, suggests that polynucleotide 5'-triphosphatase is an "early" or prereplicative viral protein. Immunologlobulin produced against the purified virion-associated polynucleotide 5'-triphosphatase as antigen neutralized the activity of the induced polynucleotide 5'-triphosphatase, thus indicating the identity of the two enzymes.

Herpesvirus transcription: Altered regulation induced by FUdR

The effect of inhibition of viral DNA replication on the regulated production of specific classes of viral transcripts was examined in equine herpesvirus type 1 infected L-M cells. 5-Fluoro-2-deoxyuridine (FUdR) was shown to inhibit viral DNA synthesis and to alter the synthesis of the two classes of viral transcripts that differ in abundance (molar concentration) [Cohen, J. C., Randall, C. C., and O'Callaghan, D. J., Virology 68, 561–565 (1975)]. In uninhibited infection, the ratio of the molar concentration of the major RNA class (most abundant) to minor RNA class (less abundant) was regulated by initiation of viral DNA synthesis since a ratio of 82.7 to 1 for “early” infection (before viral DNA synthesis) decreased to 24 to 1 for “late” infection (after initiation of viral DNA synthesis). Studies employing both standard and summation Scatchard analyses of RNA-DNA hybridization saturation data as well as RNA competition hybridization analysis revealed that in the presence of FUdR viral transcripts comprising the minor RNA class were synthesized in large amounts as compared to uninhibited infection and that sequences usually not transcribed until late were synthesized early in the FUdR-inhibited infection. In addition, it was found that the synthesis of a significant portion of transcripts that comprise the major class of uninhibited infection was blocked in the FUdR-inhibited infection. The ratios of molar concentration of major to minor RNA class present in the FUdR-inhibited infection was found to be 13.9 and 9.2 for early and late times of the infection; these low ratios (as compared to uninhibited infection) and the data of summation Scatchard analysis indicate that reiterated synthesis of certain minor class sequences occurs prematurely (early instead of late) in the presence of FUdR. These findings reveal the importance of the initiation of viral DNA synthesis in the regulation and programming of herpesvirus transcription.

The control of SV40 transcription during a lytic infection: late RNA synthesis in the presence of inhibitors of DNA replication

The transition from early to late transcription of SV40 DNA in productively infected BSC-1 cells was analyzed using both inhibitors of DNA replication, and early (Group A) temperature sensitive (ts) mutants of SV40. Late virus specific cytoplasmic RNA sedimenting at 16S in neutral sucrose gradients and complementary to the plus (L) DNA strand of SV40 was detected in cultures infected in the presence of three inhibitors of DNA replication (Ara-C, FdU, and chloroquine), even though the inhibition of viral DNA replication appeared to be essentially complete. After infection with the early SV40 mutant tsA58, no DNA replication was detected at the restrictive temperature (41 degrees C) and no significant late RNA complementary to the plus (L) strand was found, in either the cytoplasm or nuclei of infected cells. These data support the concept that expression of late viral functions requires the initiation of viral DNA synthesis or a functional gene A protein, or both.

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.