Functional relevance of specific interactions between herpes simplex virus type 1 ICP4 and sequences from the promoter-regulatory domain of the viral thymidine kinase gene

Abstract

The herpes simplex virus (HSV) type 1 immediate-early regulatory protein ICP4 is required for induced expression of HSV early and late genes, yet the mechanism by which this occurs is not known. We examined the promoter and flanking sequences of the HSV early gene that encodes thymidine kinase for the ability to interact specifically with ICP4 in gel retardation assays. Protein-DNA complexes containing ICP4 were observed with several distinct regions flanking the tk promoter. cis-Acting elements that interact with cellular transcription factors were apparently not required for these interactions to form. Purified ICP4 formed protein-DNA complexes with fragments from these regions, and Southwestern (DNA-protein blot) analysis indicated that the interaction between ICP4 and these sequences can be direct. None of the tk sequences that interact with ICP4 contains a consensus binding site for ICP4 (S. W. Faber and K. W. Wilcox, Nucleic Acids Res. 14:6067-6083, 1986), reflecting the ability of ICP4 to interact with more than one DNA sequence. A mutated ICP4 protein expressed from the viral genome that retains the ability to bind to a consensus binding site but does not bind specifically to the identified sites flanking the tk promoter results in induced transcription of the tk gene. These data support hypotheses for ICP4-mediated transactivation of the tk promoter in Vero cells that do not require the intrinsic ability of ICP4 to bind specifically in or near the promoter of the tk gene.

Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication

“…ICP4 is a phosphorylated DNA-binding protein with an apparent molecular mass of 175 kDa (6,14,32,46,63). It can bind specifically to DNA, and its binding site is relatively degenerate (14,16,26,28,32,33,(38)(39)(40). ICP4 also appears as three electrophoretic species on sodium dodecyl sulfate (SDS)polyacrylamide gels (46) and as seven species on two-dimensional isoelectrofocusing gels (1).…”

Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences

“…The expression levels of proteins in cell lines and primary cells or tissues of organisms vary and are differentially controlled due to cell-specific expression of transcription factors and their posttranslational modifications as well as additional factors such as small noncoding RNA [32][33][34][35][36][37][38][39][40]. For cell biological experiments, it has become of interest to achieve precise expression levels for recombinant proteins in cells [41,42].…”

“…For cell biological experiments, it has become of interest to achieve precise expression levels for recombinant proteins in cells [41,42]. The strength of the well-established herpes simplex virus (HSV) thymidine kinase (TK) promoter has been shown to be in general weaker than that of other widely used viral promoters, the strong CMV and SV40 promoters [33,34,38,42,43]. Studies have shown that the TK promoter contains a TATA box, a CCAAT box, and two SP1 elements ( [32][33][34][35][44][45][46] reviewed in [47]).…”

“…The strength of the well-established herpes simplex virus (HSV) thymidine kinase (TK) promoter has been shown to be in general weaker than that of other widely used viral promoters, the strong CMV and SV40 promoters [33,34,38,42,43]. Studies have shown that the TK promoter contains a TATA box, a CCAAT box, and two SP1 elements ( [32][33][34][35][44][45][46] reviewed in [47]). They are important to modulate the expression of viral genes and are bound by cellular transcription factors [32][33][34][35][36][37][38][39][40].…”

“…Studies have shown that the TK promoter contains a TATA box, a CCAAT box, and two SP1 elements ( [32][33][34][35][44][45][46] reviewed in [47]). They are important to modulate the expression of viral genes and are bound by cellular transcription factors [32][33][34][35][36][37][38][39][40]. In addition, the region of the 5 0 untranslated region (5 0 -UTR) of the HSV TK gene may also be involved in the regulation of transcription of the TK gene ( [33,34,46,48] summarized in [47]).…”