Mutational Analysis of the Herpes Simplex Virus Type 1 Trans-inducing Factor Vmw65

Abstract

SUMMARYThe herpes simplex virus type 1 (HSV-1) polypeptide Vmw65 is a structural component of the virus particle and is also responsible for trans-induction of immediate early (IE) transcription. Functional domains of this polypeptide were investigated by constructing a series of 10 plasmids each with a 12 bp insertion in the gene encoding Vmw65. Plasmids were analysed for their ability to stimulate IE transcription in short term transfection assays, and the altered Vmw65 polypeptides were assayed for the ability to form an IE-specific protein-DNA complex (IEC) in vitro. A direct correlation was observed between stimulation of transcription and formation of IEC, strongly suggesting that IEC is an important intermediate in transcription activation. Plasmids were also tested for their ability to rescue the temperature-sensitive mutation in the HSV-2 assembly mutant ts2203, since marker rescue analysis indicated that this mutation maps within the gene encoding Vmw65. Five plasmids failed to rescue ts2203, thereby defining regions of Vmw65 required for virus assembly. The results show that distinct domains exist in Vmw65 for activation of transcription and assembly of virus.

The herpes simplex virus trans-activator VP16 recognizes the Oct-1 homeo domain: evidence for a homeo domain recognition subdomain.

“…The structures of the four mutant VP16 proteins are shown in Figure 2, which also summarizes the results of these experiments. Previous mutagenesis studies (Ace et al 1988;Werstuck and Capone 1989a, b;O'Hare 1989, 1990) have identified two important regions for VP 16-induced complex formation; we refer to these as region I (amino acids 173-241) and region II (amino acids 317-403). We constructed two mutants, RI-A and RII-A, in which regions I and II are deleted individually.…”

“…We constructed two mutants, RI-A and RII-A, in which regions I and II are deleted individually. We also assayed the activities of two previously characterized 4-amino-acid insertion mutants, in9 and inl4 (Ace et al 1988), which we refer to here simply as RI-i and RII-i, respectively. These two mutations disrupt formation of the HCF-dependent VP16-induced complex; the RII-i mutant, however, can still participate in virion assembly, suggesting that its structure is not grossly altered (Ace et al 1988).…”

“…Two deletions were individually made in wild-type VP16: either region I, extending from amino acid 173 to 241 inclusive (RI-A), or region II, extending from amino acid 317 to 403 inclusive (RII-A), was deleted. In addition, the previously described 4-amino-acid insertion mutations, in9 (after amino acid 177) and inl4 (after amino acid 379) (Ace et al 1988), were introduced individually within regions I and II and are referred to here as RI-i and RII-i, respectively. The amino acids inserted by the in9 and inl4 mutations are indicated below their respective positions.…”

Establishment of latency in vitro by the herpes simplex virus type 1 mutant in 1814

“…The probe was a radiolabelled 1.6 kbp HindlII/EcoRI fragment from pMC 17 (Ace et al, 1988), containing the coding sequences for Vmw65.…”

Amino Acid Substitutions in the Herpes Simplex Virus Transactivator VP16 Uncouple Direct Protein-Protein Interaction and DNA Binding from Complex Assembly and Transactivation

“…VP16 is a modular protein and contains separable domains that are important for complex formation and transactivation (19,(23)(24)(25)(26)(27)(28)(29). The carboxyl-terminal acidic activation domain is essential for transcriptional activation but not for complex assembly.…”