Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression

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Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a K m within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.

Section: Methodsmentioning

confidence: 99%

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

Xia

Knipe

DeLuca

1996

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“…, and E26 were maintained as previously described (4,10). E5 cells were previously described (5), and E8 and E26 cells are described herein.…”

Section: Virus and Cells Vero Cells And The Complementing Cell Linesmentioning

confidence: 99%

“…ICP4 is a 175-kDa phosphoprotein (3,47) that directly acts as a transcriptional regulatory protein. It is required for the induction of early and late genes and acts as a repressor of its own expression and that of several other viral genes (4,6,10,43,44,49). The ICP4 protein consists of several modular domains that encode specific functions including those for DNA binding, nuclear localization, dimerization, and transcriptional activation (8,45,46,66).…”

mentioning

confidence: 99%

“…The ICP4 protein consists of several modular domains that encode specific functions including those for DNA binding, nuclear localization, dimerization, and transcriptional activation (8,45,46,66). The protein has sequence-specific DNA-binding ability, with preference for the consensus ATCGTCN 4 YGCRC present in its own promoter as well as other HSV-1 gene promoters (13,30,38,40). ICP4 represses promoters containing a strong ICP4 binding site located near the start site of transcription (6,20,37,44,55).…”

mentioning

confidence: 99%

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Functional interactions between herpes simplex virus immediate-early proteins during infection: gene expression as a consequence of ICP27 and different domains of ICP4

Samaniego

Webb

DeLuca

1995

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119

Two of the five immediate-early gene products, ICP4 and ICP27, expressed by herpes simplex virus type 1 have profound effects on viral gene expression and are absolutely essential for virus replication. Functional interactions between ICP4 and ICP27 may contribute to establishing the program of viral gene expression that ensues during lytic infection. To evaluate this possibility, viral mutants simultaneously deleted for ICP27 and defined functional domains of ICP4 were constructed. These mutant viruses allowed a comparison of gene expression as a function of different domains of ICP4 in the presence and absence of ICP27. Gene expression in the absence of both ICP4 and ICP27 was also examined. The results of this study demonstrate a clear involvement for ICP27 in the induction of early genes, in addition to its known role in enhancing late gene expression during viral infection. In the absence of both ICP4 and ICP27, viral early gene expression, as measured by the accumulation of thymidine kinase and ICP6 messages was dramatically reduced relative to the amounts of these messages seen in the absence of only ICP4. Therefore, elevated levels of early gene expression as a consequence of ICP27 occurred in the absence of any ICP4 activity. Evidence is also presented regarding the modulation of the ICP4 repression function by ICP27. When synthesized in the absence of ICP27, a mutant ICP4 protein was impaired in its ability to repress transcription from the L/ST promoter in the context of viral infection and in vitro. This defect correlated with the loss of the ability of this mutant protein to bind to its recognition sequence when produced in infected cells in the absence of ICP27. These observations indicate that ICP27 can regulate the activity of at least one domain of the ICP4 protein as well as contribute to elevated early gene expression independently of ICP4.

“…ICP4 (or Vmw175) is one of five immediate-early proteins of herpes simplex virus type 1 (HSV-1) and plays an essential role in regulating the expression of all three kinetic classes of HSV-1 genes (5-7, 10, 19, 22). Temperature-sensitive and deletion mutants of ICP4 are not viable in cells cultured at nonpermissive conditions (5,7,8,22). The function of ICP4 has been shown to (i) activate the transcription of early and late genes, (ii) induce viral DNA synthesis, and (iii) down regulate immediate-early gene expression (5,7).…”

mentioning

confidence: 99%

Initial characterization of the membrane-associated form of ICP4 of herpes simplex virus type 1

Xia

Courtney

1995

The immediate-early gene product, ICP4, of herpes simplex virus type 1 (HSV-1) is one of the major transcriptional regulatory proteins in the virus replicative process and is localized primarily within the nucleus soon after its synthesis. Earlier studies have shown that detectable amounts of ICP4 are also associated with the plasma membranes of infected cells (F. Yao and R. J. Courtney, J. Virol. 65:1516-1524, 1991). To extend our understanding of the properties of the membrane-associated ICP4, we have used various electrophoretic techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and isoelectric focusing, to compare the membrane-and nuclear-associated forms of ICP4. The data from all of these methods revealed that a single unique form of ICP4 associates with plasma membranes of HSV-1-infected cells. While multiple forms of ICP4 were detected in infected cell nuclei, the membraneassociated form of ICP4 appeared to have a lower apparent molecular weight and a more acidic pI than the various forms of ICP4 found in infected cell nuclei. These results suggest that a novel form of ICP4 may associate with plasma membranes of HSV-1-infected cells. A recombinant adenovirus, AdICP4 (encoding an ICP4 protein), was used to determine the role that other herpesvirus proteins may play in the membrane association of ICP4. The results suggest that the expression of other HSV-1 proteins is not required for the membrane association of ICP4.