This phosphorylation modification generates a novel form of the large subunit which we have designated IIi. In this study, we examine the roles that HSV-1 gene products play in this process. An HSV-1 mutant defective in the immediate-early transcriptional activator protein ICP4 is able to efficiently induce IIi. Viruses having mutations in the genes for the ICP0, ICP6, or ICP27 proteins are also competent for IIi formation. In contrast, 22/n199, an HSV-1 mutant which contains a nonsense mutation in the gene encoding the immediate-early protein ICP22, is significantly deficient in IIi induction. This effect is seen in Vero cells, where 22/n199 grows relatively efficiently, and in human embryonic lung (HEL) cells, where 22/n199 growth is more restricted. RNAP II is recruited into viral replication compartments in 22/n199-infected cells, indicating that altered phosphorylation of RNAP II is not a prerequisite for nuclear relocalization of RNAP II. In addition, we show by nuclear run-on transcription analysis that viral gene transcription is deficient in HEL cells infected with 22/n199. Viral late gene transcription does not occur efficiently, and antisense transcription throughout the genome is diminished compared with that of the wild-type HSV-1 infection. These transcriptional effects cannot be explained by differences in viral DNA replication, since 22/n199 replicates its DNA efficiently in HEL cells. Our results demonstrate that ICP22 is necessary for virus-induced aberrant phosphorylation of RNAP II and for normal patterns of viral gene transcription in certain cell lines.Herpes simplex virus type 1 (HSV-1), a common human herpesvirus, provides a useful model to study various aspects of herpesvirus gene expression and regulation (for a review, see reference 47). Like all herpesviruses, HSV-1 replicates in the nucleus of its host cell, and to a large extent depends on the cell's synthetic machinery for the expression of its genes. Transcription of the viral genes is mediated by host RNA polymerase II (RNAP II). The genome of HSV-1 is a linear double-stranded DNA molecule, ϳ152 kb in size, encoding approximately 75 genes. For the most part, the HSV-1 genes are similar in structure to cellular genes, possessing typical eukaryotic promoter and polyadenylation sequences. During lytic infection, the viral genes are induced to high levels, while expression of cellular genes is greatly suppressed (for a review, see reference 56). Moreover, HSV-1 gene expression is tightly controlled. The viral genes have been grouped into three classes on the basis of the temporal order of, and requirements for, their expression (24). The first genes to be expressed are the immediate-early (IE; also called ␣) genes. Their expression does not require newly synthesized viral proteins but does require the action of the virion component VP16. The second class of genes are the delayed-early (DE; also called ) genes. The expression of DE (and later) genes is dependent upon newly synthesized IE gene products, in particular the ICP4 protein, ...
During lytic infection, herpes simplex virus subverts the host cell RNA polymerase II transcription machinery to efficiently express its own genome while repressing the expression of most cellular genes. The mechanism by which RNA polymerase II is directed to the viral delayed-early and late genes is still unresolved. We report here that RNA polymerase II is preferentally localized to viral replication compartments early after infection with herpes simplex virus type 1. Concurrent with recruitment of RNA polymerase II into viral compartments is a rapid and aberrant phosphorylation of the large subunit carboxy-terminal domain (CTD). Aberrant phosphorylation of the CTD requires early viral gene expression but is not dependent on viral DNA replication or on the formation of viral replication compartments. Localization of RNA polymerase II and modifications to the CTD may be instrumental in favoring transcription of viral genes and repressing specific transcription of cellular genes.
The DNA-dependent protein kinase (DNA-PK) is involved in several fundamental nuclear processes, including DNA double-strand break repair, V(D)J recombination, and transcription by RNA polymerases I and II. In this study, we show that infection of mammalian cells with herpes simplex virus type 1 attenuates DNA-PK activity by specifically depleting the p350/DNA-PKcs catalytic subunit. The half-life of the p350/DNA-PKcs protein decreases from greater than 24 h to less than 4 h following infection. The depletion of DNA-PK activity and p350/DNA-PKcs abundance is dependent on expression of the viral immediate-early protein ICP0. As ICP0 acts as a promoter-independent transactivator of gene expression, these data suggest that ICP0 may function by directly or indirectly targeting the p350/DNA-PKcs subunit of DNA-PK, thereby altering the inhibitory effects of DNA-PK on RNA polymerase II transcription.
Leaf spots and root rots are major fungal diseases in Camptotheca acuminata that limit cultivation of the plant for camptothecin (CPT), a promising anticancer and antiviral alkaloid. Bioassays showed that pure CPT and flavonoids (trifolin and hyperoside) isolated from Camptotheca effectively control fungal pathogens in vitro, including Alternaria alternata, Epicoccum nigrum, Pestalotia guepinii, Drechslera sp., and Fusarium avenaceum, although antifungal activity of these compounds in the plant is limited. CPT inhibited mycelial growth by approximately 50% (EC50) at 10-30 microg/mL and fully inhibited growth at 75-125 microg/mL. The flavonoids were less effective than CPT at 50 microg/mL, particularly within 20 days after treatment, but more effective at 100 or 150 microg/mL. CPT, trifolin, and hyperoside may serve as leads for the development of fungicides.
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