Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.     

Inhibition of the synthesis of proteins needed for Epstein-Barr virus replication by antisense RNA against the zta gene

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication.     

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).     

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.     

Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid.

Phosphonoacetic acid specifically inhibited human cytomegalovirus DNA synthesis in virus-infected human fibroblasts as detected by virus-specific nucleic acid hybridization. Inhibition was reversible; viral DNA synthesis resumed upon the removal of the drug. The compound partially inhibited DNA synthesis of host cells in the log phase of growth but had little effect on confluent cells. Studies of partially purified enzymes indicated that phosphonoacetic acid specifically inhibited virus-induced DNA polymerase and had only a slight effect on normal host cell enzymes. The drug was shown to interact directly with virus-induced enzyme but not with the template-primers.     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

Epstein-Barr virus DNA. X. Direct repeat within the internal direct repeat of Epstein-Barr virus DNA.

The 3,360-base-pair internal direct repeat (IR) in Epstein-Barr virus DNA separates the short and long unique DNA domains. IR has a single BamHI site. The juncture between the short unique domain and IR has been mapped by restriction endonucleases and is less than 2,600 nucleotides before the BamHI site in IR. The junction between IR and the long unique domain has been sequenced and is approximately 650 nucleotides after the BamHI site in IR. Thus, relative to the start of IR at the juncture with the short unique domain, the last repeat is at least 90 base pairs short of being complete. There is homology between the 250-nucleotide fragments to the left and the right of the unique BamHI site in IR. A 35-base-pair sequence of the left fragment is directly repeated within the right fragment, once fully and once partially. The implications of these findings are discussed.     

Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.     

Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling.

Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged. Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level. Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling.     

Aurintricarboxylic acid (ATA) and DNA synthesis. I. Inhibition of DNA synthesis by ATA in Go cells stimulated to proliferate.

Aurintricarboxylic acid (ATA) at a concentration which produces 40% inhibition of protein synthesis, inhibits completely isoproterenol-stimulated DNA synthesis in mouse parotid glands. The drug was found to interfere with some essential changes occurring during the prereplicative phase of IPR-stimulated DNA synthesis. It inhibits the increase in ribosonal protein synthesis that takes place by 2 h after stimulation. The peak of ribosonal RNA that occurs 8 h after isoproterenol was also abolished by ATA. Since the drug completely inhibits isoproterenol-stimulated DNA synthesis, these results suggest that the control of ribosome production may be involved in cell growth activation. In view of the finding that ATA first inerferes with the binding of adenylate-rich RNA to polysomes, it was suggested that the drug may act by preferentially inhibiting that fraction of protein synthesis dependent on the newly transcribed messenger RNA.     

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.     

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved or bended helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.