Nucleotides within both proximal and distal parts of the consensus sequence are important for specific DNA recognition by the herpes simplex virus regulatory protein ICP4

Pizer, Lewis I.; Everett, Roger D.; Tedder, Davol G.; Elliott, Margaret; Litman, Burton J.

doi:10.1093/nar/19.3.477

Cited by 13 publications

(31 citation statements)

References 29 publications

(30 reference statements)

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“…Cloning and expression of the DNA binding domain of 140k in a T7 expression vector and its subsequent partial purification Gene 62, encoding the 140k protein, lies in the left repeat bounding the short unique segment of the VZV genome ( ;46 417I 546 417 expressed in isolation and found to have a similar binding specificity to that of the intact protein (21,25,31). Our intention was to express the equivalent region of the homologous VZV JE protein 140k, encompassing its DNA binding domain, in order to study its DNA binding characteristics for comparison with those of the HSV-1 Vmw175 DNA binding domain.…”

Section: Resultsmentioning

confidence: 99%

The DNA binding domain of the Varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1

Tyler¹,

Everett

1993

Nucl Acids Res

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Varicella-zoster virus gene 62 encodes a protein with predicted Mr of 140,000D (VZV 140k) that shares extensive predicted amino acid sequence homology with the major immediate early (IE) transcriptional regulator protein of herpes simplex virus type 1 (HSV-1) Vmw175. The integrity of highly conserved region 2 is essential for the DNA binding and transcriptional regulatory functions of Vmw175. Similarly, an insertion mutation in region 2 (codons 468-641) of 140k eliminates the transcriptional repression and activation functions of this protein. We have expressed a fragment of 140k which encompasses region 2 as a non-fusion polypeptide in bacteria. This 140k DNA binding domain peptide (codons 417-646) binds to numerous DNA sequences throughout the VZV gene 62 promoter region. It induces multiple regions of protection from DNase I digestion, flanked by sites of DNase I hypersensitivity. Several of the sites recognized can be considered to be divergent forms of the consensus sequence which is recognized by Vmw175. However, by use of a panel of mutagenized probe fragments, we found that the 140k DNA binding domain was less sequence-specific than Vmw175 in its interactions with DNA. Consistent with this, the homologous Vmw175 DNA binding domain, and also intact Vmw175, recognize the gene 62 binding sites much less efficiently than the 140k DNA binding domain. Also in contrast to the situation with Vmw175, the 140k DNA binding domain failed to induce DNA bending when occupying the binding sites in its own promoter. Deletion analysis has mapped the minimal DNA binding domain of the VZV 140k protein, as measured in gel retardation analysis, to lie within residues 472 to 633. The differences in binding characteristics of the DNA binding domains of the homologous VZV 140k and HSV-1 Vmw175 IE proteins may account for the subtle differences in their regulatory activities in transfection assays and during virus growth in tissue culture.

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Section: Resultsmentioning

confidence: 99%

The DNA binding domain of the Varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1

Tyler¹,

Everett

1993

Nucl Acids Res

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“…l a). A consensus IE175K recognition site of the form ATCGTCnnnnyC/G G (n is any base) has been proposed in several studies (Faber & Wilcox, 1988;DiDonato et al, 1991;Pizer et al, 1991). Two potential IE175K binding sites in LAP, which are similar to the consensus and form an imperfect inverted palindrome, are indicated by dashed arrows (Fig.…”

Section: Binding Of Le175k To Lap Cap Site Sequencesmentioning

confidence: 99%

“…The results of DeLuca & Schaffer (1988) indicated that large regions of IE175K could be deleted without affecting DNA binding. Moreover, small recombinant IE175K variants expressed in E. coli were sufficient for DNA binding and for dimerization (Wu & Wilcox, 1990;Everett et al, 1991;Pizer et at., 1991). Bacterial fusion proteins FP449 and FP525, containing IE175K residues 262 to 490 that span the DNA-binding domain (Wu & Wilcox, 1990), were therefore tested to determine whether they retained the capacity to form complex C2.…”

Section: Protein Requirements For C2 Formationmentioning

confidence: 99%

“…nature, a n d contains a D N A -b i n d i n g d o m a i n in addition to regions specifically required for trans-activation (Muller, 1987;DeLuca & Schaffer, 1988;F a b e r & Wilcox, 1988;Paterson & Everett, 1988a, b;Shepard et al, 1989;W u & Wilcox, 1990;Everett et al, 1991 ;Pizer et al, 1991). IE175K binds to D N A sequences that c o n t a i n the consensus core m o t i f A T C G T C ( F a b e r & Wilcox, 1988;D i D o n a t o et al, 1991;Everett et al, 1991), b u t also binds to a variety of sequences that have diverged from the consensus (Michael et al, 1988;Tedder & Pizer, 1988;Michael & Roizman, 1989; I m b a l z a n o et al D i D o n a t o et al, 1991;Papavassiliou et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein

Batchelor

Wilcox

O’Hare

1994

Journal of General Virology

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We demonstrate that the immediate early 175K protein (IE175K) of herpes simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP cap site was significantly larger than that formed on the IE175K cap site and the requirements for binding were qualitatively distinct with respect to both the prirnary sequence determinants at the site, and the regions of IE 175K protein required for binding compared to those for the IE175K cap site. Although purified IE175K was sufficient for this larger complex formed on the LAP cap site, the DNA-binding domain was unable to bind efficiently. This contrasted strikingly with the IE175K cap site where, using precisely analogous probes, the DNA-binding domain exhibited a strong interaction. Surprisingly, from dissociation kinetics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap site (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expression than IEI75K promoter-driven expression by IEI75K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region differed from previously identified IE175K recognition sequences in that in addition to a partially conserved consensus sequence, neighbouring bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to this region. Together these results indicate that IE175K forms an unusual complex at the LAP cap site, broadening the range of previously defined sequences recognized by IE175K.

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“…Plasmids pT7I9, pT7I9X, pT7I10 and pT7I10X, which express variations of the DNA binding domain of Vmw175 from the p585.4 vector (based on pET8c), have been described previously Pizer et al, 1991). Plasmids were constructed and maintained in E. coli strain DH5 and expression was induced in strain BL21(DE3)pLysS (Studier et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

Mutations which alter the DNA binding properties of the herpes simplex virus type 1 transactivating protein Vmw175 also affect its ability to support virus replication.

Allen¹,

Everett²

1997

Journal of General Virology

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The crucial role of herpes simplex virus type 1 immediate early protein Vmw175 (ICP4) in the regulation of all classes of viral genes has been established by extensive analysis of temperaturesensitive, insertion and deletion mutants. It has long been known that Vmw175 binds to selected DNA sequences, and recent studies have shown that it interacts with components of the basal transcription machinery. However, the role of DNA binding in its mechanism of action has been controversial. Despite the presence of Vmw175 recognition sites at numerous locations throughout the viral genome, it has proved difficult to establish that these sites are

show abstract

Nucleotides within both proximal and distal parts of the consensus sequence are important for specific DNA recognition by the herpes simplex virus regulatory protein ICP4

Cited by 13 publications

References 29 publications

The DNA binding domain of the Varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1

The DNA binding domain of the Varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1

Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein

Mutations which alter the DNA binding properties of the herpes simplex virus type 1 transactivating protein Vmw175 also affect its ability to support virus replication.

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