Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression.

Abstract

Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16. The action of VP16 is mediated through cis-regulatory elements located in regions adjacent to each IE gene. Although VP16 is normally introduced into cells by infecting virions, its trans-activating function can also be observed by cotransfecting cells with a plasmid that encodes VP16 along with a reporter gene driven by IE cisregulatory sequences. We have used such an assay to examine the function of mutant forms of VP16. Our results provide tentative identification of two domains of VP16 that are crucial to its role in the induction of IE gene expression. One domain is located within the carboxy-terminal 78 amino acids of VP16 and is characterized by its acidity. Another domain, located in a more amino-terminal region of the protein, appears to tailor the specificity of VP16 for IE genes. According to the results presented in this and the accompanying paper, we predict that VP16 achieves IE gene specificity via protein : protein, rather than protein : DNA, interaction.

Chromosomal instability by β-catenin/TCF transcription in APC or β-catenin mutant cells

“…In this cell line, the Wnt transcription is relatively lower than those in other cells with Wnt signal activation (van de Wetering et al, 2002). Thus, we induced a dominant-active TCF4 (TCF4-VP16 where human TCF4 was fused with the transactivation domain of Herpes simplex virus-VP16; Triezenberg et al, 1988) in these cells, using a doxycyclin-inducible TetOff system (Materials and methods). Transcriptional activation was confirmed using Topflash and Fopflash reporters.…”

Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation

“…The twohybrid selection was performed essentially as described in Hollenberg et al (1995) with the S. cerevisiae strain L40 (MATa trp1 leu2 his3 LYS2::(lexA) 4 -HIS3 URA3::(lexA) 8 -lacZ) (Vojtek et al, 1993). L40 cells containing the pLexA ± E7(42 ± 98) target plasmid were transformed with a cDNA library derived from 9.5 ± 10.5 days post-coitus CD1 mouse embryos fused to the activation domain of Herpes simplex viral protein 16 (Triezenberg et al, 1988;Hollenberg et al, 1995), to give 6610 6 primary transformants. To select for strongly interacting proteins and to decrease background, 25 mM aminotriazol was included in the selective medium.…”

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

“…Accordingly, many approaches such as knockdown with synthesized morpholinos, insertional mutagenesis, ectopic expression of a target gene, dominantnegative and conditional gene expression, have been developed to elucidate gene functions during embryonic development. Several inducible gene expression systems including the ecdysone-regulated gene switch (Triezenberg et al, 1988), the lac operator-repressor system (Cronin et al, 2001), the inducible GAL4/UAS system (Lycett et al, 2004) and two tetracycline (Tet)-controlled systems (Gossen and Bujard, 1992;Gossen et al, 1995) have been developed for manipulating gene expression at the transcriptional level in Drosophila, mammalian cells and transgenic animals with varying degrees of success, but their activities in zebrafish are not yet well-characterized. In fact, only a limited number of transgenic zebrafish lines carrying these gene expression systems are currently available.…”

“…In the Tet-off system, a eukaryotic transcriptional activator is created by fusing amino acids 1e207 of the Tet repressor (TetR) to the C-terminal 127 amino acids of the herpes simplex virus VP16 activation domain (Triezenberg et al, 1988). Additionally, a Tet-response promoter is generated by insertion of seven Tet operator (TetO) repeat sequences just upstream of the minimal CMV promoter.…”