Repression of activator-mediated transcription by herpes simplex virus ICP4 via a mechanism involving interactions with the basal transcription factors TATA-binding protein and TFIIB.

Infected-cell polypeptide 4 (ICP4) of herpes simplex virus is both a transcriptional activator and a repressor. It has been previously demonstrated that both SP1-activated transcription and USF-activated transcription are repressed by ICP4 without affecting basal transcription (B. Gu, R. Rivera-Gonzalez, C. A. Smith, and N. A. DeLuca, Proc. Natl. Acad. Sci. USA 90:9528-9532, 1993; R. Rivera-Gonzalez, A. N. Imbalzano, B. Gu, and N.A. DeLuca, Virology 202:550-564, 1994). In this study, it was found that ICP4 repressed the activation function of two other activators, VP16 and ICP4 itself, in vitro. ICP4 inhibited transcription by interfering with the formation of transcription initiation complexes without affecting transcription elongation. Repression of activator function required that an ICP4 DNA binding site was present in one orientation within approximately 45 bp 3' to the TATA box. DNA binding by ICP4 was necessary but not sufficient for repression. ICP4 has been shown to form tripartite complexes cooperatively with the TATA box-binding protein and TFIIB on DNA containing an ICP4 binding site and a TATA box (C. A. Smith, P. Bates, R. Rivera-Gonzalez, B. Gu, and N. DeLuca, J. Virol. 67:4676-4687, 1993). A region of ICP4 that enables the molecule to form tripartite complexes was also required in addition to the DNA binding domain for efficient repression. Moreover, repression was observed only when the ICP4 binding site was in a position that resulted in the formation of tripartite complexes. Together, the data suggest that ICP4 represses transcription by binding to DNA in a precise way so that it may interact with the basal transcription complex and inhibit some general step involved in the function of activators. The steps or interactions involved in transcriptional activation that are inhibited by ICP4 are discussed.

Interaction of the viral activator protein ICP4 with TFIID through TAF250.

ICP4 of herpes simplex virus is responsible for the activation of viral transcription during infection. It also efficiently activates and represses transcription in vitro depending on the promoter context. The contacts made between ICP4 and the cellular proteins that result in activated transcription have not been identified. The inability of ICP4 to activate transcription with TATA-binding protein in place of TFIID and the requirement for an initiator element for efficient ICP-4-activated transcription suggest that coactivators, such as TBP-associated factors, are involved (B. Gu and N. DeLuca, J. Virol. 68:7953-7965, 1994). In this study we showed that ICP4 activates transcription in vitro using an immunopurified TFIID, indicating that TBP-associated factors may be a sufficient subset of coactivators for ICP4-activated transcription. Similar to results seen in vivo, the presence of the ICP4 C-terminal region (amino acids 774 to 1298) was important for activation in vitro. With epitope-tagged ICP4 molecules in immunoaffinity experiments, it was shown that the C-terminal region was also required for ICP4 to interact with TFIID present in a crude transcription factor fraction. In the same assay, ICP4 was unable to interact with the basal transcription factors, TFIIB, TFIIE, TFIIF, and TFIIH and RNA polymerase II. ICP4 could also interact with TBP, independent of the C-terminal region. However, reflective of the interaction between ICP4 and TFIID, the ICP4 C-terminal region was required for an interaction with FAF250-TBP complexes and with TAF250 alone. Therefore, the interfaces or conformation of TBP mediating the interaction between ICP4 and TBP in solution is probably masked when TBP is bound to TAF250. With a series of mutant ICP4 molecules purified from herpes simplex virus-infected cells, it was shown that ICP4 molecules that can bind DNA and interact with TAF250 could activate transcription. Taken together, these results demonstrate that ICP4 interaction with TFIID involves the TAF250 molecule and the C-terminal region of ICP4 and that this interaction is part of the mechanism by which ICP4 activates transcription.

Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4.

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.

Induction of transcription by a viral regulatory protein depends on the relative strengths of functional TATA boxes.

The mechanisms by which viral regulatory proteins activate the cellular transcription apparatus without binding to specific DNA elements are not fully understood. Several lines of evidence suggest that activation by one such regulatory protein, herpes simplex virus ICP4, could be mediated, at least in part, by TFIID. To test this model, we replaced the TATA box of the ICP4-responsive viral thymidine kinase gene with functional TATA boxes that displayed different apparent affinities for TATA-box-binding protein as measured by DNase I footprinting. We measured the effects of these TATA boxes on ICP4 induction by constructing ICP4-deficient recombinant viruses containing the different TATA alleles and comparing their expression in cells lacking or expressing ICP4. Overall, ICP4 induced weak TATA boxes (those that displayed low apparent affinity for TATA-box-binding protein and low basal expression) the most (18- to 41-fold) and strong TATA boxes the least (7- to 10-fold). Therefore, ICP4 induction correlated inversely with TATA box strength. Using a reconstituted in vitro transcription assay, we determined that the relative levels of induction by ICP4 of the different TATA alleles were similar to those measured in vivo, suggesting that ICP4 was the only viral protein required for induction. These results fit a model in which ICP4 acts in part to enhance binding of TFIID to the TATA box. We compare and contrast these results with those observed with the viral regulatory proteins adenovirus E1a and simian virus 40 large T antigen and the cellular coactivator PC4.

Activities of heterodimers composed of DNA-binding- and transactivation-deficient subunits of the herpes simplex virus regulatory protein ICP4.

Two mutant strains (vi12 and vi13) of herpes simplex virus that contain insertion mutations in the sequences that encode the DNA-binding domain of viral regulatory protein ICP4 were generated. Both mutations disrupted specific DNA binding and resulted in transcriptionally inactive molecules; however, the ability of the mutant proteins to form dimers was retained. The mutant proteins formed heterodimers with an ICP4 deletion mutant (X25) that is nonfunctional but retains the ability to bind to consensus sites. Significantly elevated levels of early (E or beta) and "leaky late" (beta gamma or gamma 1) gene expression were observed upon coexpression of the insertion mutant and X25 ICP4 polypeptides. While the heterodimers composed of the vi13 and X25 peptides possessed DNA-binding activity, those composed of vi12 and X25 did not, indicating that DNA binding by the heterodimers may not be required for restored activity. Despite significant levels of early gene expression and viral DNA synthesis in vi12-infected X25 cells, true late (gamma 2) mRNA was not synthesized. This indicates that the structural requirements for ICP4 induction of different classes of viral genes may be different.

Intragenic complementation among partial peptides of herpes simplex virus regulatory protein ICP4.

Peptides of the herpes simplex virus type 1 regulatory protein, ICP4, which are translated from genes containing nonsense and deletion mutations retain specific biochemical properties and activities characteristic of the intact ICP4 molecule (N. A. DeLuca and P. A. Schaffer, J. Virol. 62:732-743, 1988). Mutant viruses expressing these peptides are deficient for viral growth in the absence of complementing wild-type protein supplied in trans, indicating that the mutant peptides are not functionally complete. In the present study we have demonstrated that certain pairs of mutants expressing partial ICP4 peptides complement each other. The complementation is shown at the level of transcription and results in enhanced virus growth. Among complementing pairs of ICP4 mutants is a virus expressing a peptide deleted for codons 185 to 309 (d2) and a virus expressing only the amino-terminal 774 amino acids (n208). By using a mobility-shift assay and by taking advantage of the specific DNA-binding properties of ICP4, it was demonstrated that novel ICP4-containing DNA-protein complexes were found when extracts from cells coinfected with complementing pairs of ICP4 mutants were incubated with target DNA. The novel complexes were shown to be a function of both mutant peptides in the coinfected cell, suggesting that complementation results from the multimerization of partial ICP4 peptides.

Transdominant inhibition of herpes simplex virus growth in transgenic mice.

A mutant allele (X25) of an essential regulatory protein, ICP4, encoded by herpes simplex virus (HSV) has been shown to have a transdominant, negative effect on the activity of the wild-type protein, resulting in the inhibition of virus growth in vitro. The X25 protein appears to exert its transdominant effect by sequestering functional ICP4 monomers into nonfunctional, heterodimeric complexes (A. Shepard, P. Tolentino, and N. A. DeLuca, 1990, J. Virol. 64, 3916-3926). In order to assess the antiviral potential of X25 in vivo, four transgenic mouse lines were generated bearing 1 to 10 copies of a DNA fragment encoding the mutant allele. Monolayers of embryonic cells prepared from each of the lines expressed the transgenic X25 protein. When challenged via the eye, every line exhibited at least some enhanced resistance to HSV infection. In the best line, transgenic animals exhibited a statistically significant (> 95% confidence) 5- to 13-fold lower eye swab titer relative to their nontransgenic littermates at Day 1 postinfection. A similar reduction in titer was observed in the trigeminal ganglia at Day 3 postinfection. These results indicate that the X25 protein is able to exert a significant antiviral effect in vivo.

Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression.

The role of cis-acting promoter elements associated with herpes simplex virus type 1 (HSV-1) early and late genes was evaluated during productive infection with regard to activation of gene expression by the HSV-1 transactivator ICP4 and control of temporal regulation. A set of recombinant viruses was constructed such that expression of an HSV-1 early gene, thymidine kinase (tk), was placed under the control of either the tk TATA box or the TATA box from the late gene, glycoprotein C (gC), in the presence or absence of the upstream Sp1 and CCAAT sites normally found in the tk promoter. The presence of Sp1 sites in the promoter or replacement of the tk TATA box with the gC TATA box resulted in a decreased activation of tk mRNA expression by ICP4. Substitution of the A + T-rich region from the gC TATA box in the context of the remainder of the surrounding tk sequences resulted in a promoter that bound recombinant TATA-binding protein (TBP) better at lower concentrations than the wild-type tk promoter did. These results indicate that tk promoters that are better able to utilize TBP are less responsive to ICP4 activation and suggest that activation by ICP4 involves the general transcription factors that interact with TBP or TBP itself. Additionally, all of the viruses expressed tk at early times postinfection, indicating that cis-acting promoter elements that control the level of expression of HSV-1 early and late genes do not determine temporal regulation.

Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4.

A characteristic common to DNA animal viruses is the expression early in infection of viral proteins that act in trans to regulate subsequent RNA polymerase II-dependent transcription of the remainder of the viral genome. The predominant transcriptional regulatory protein specified by herpes simplex virus type 1 is the immediate-early protein ICP4. ICP4 is a complex multifunctional protein required for the activation of many herpes simplex virus type 1 transcriptional units and for repression of its own transcription. In the present study we have introduced nonsense and deletion mutations into both genome copies of the ICP4 gene such that the resulting mutants express only defined subsets of the primary ICP4 amino acid sequence. The partial peptides retain activities and physical properties of the intact ICP4 molecule, permitting one to attribute individual activities and properties to defined amino acid sequences.

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.

Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0.

Herpes simplex virus type 1 is capable of inhibiting host cell DNA synthesis following lytic infection. However, the mechanism and nature of potential effects on cell cycle progression have not been described. In this report, we characterize the dysregulation of the cell cycle following infection with the replication-incompetent virus d106, where immediate-early gene expression is restricted to infected-cell polypeptide 0 (ICP0) and the expression of all other viral genes is dramatically reduced or is not observed. Infection with d106 resulted in the accumulation of cells in both the G(1)/S and G(2)/M compartments, consistent with cell cycle arrest at both checkpoints. The isogenic variant d109, which does not express any viral proteins, failed to induce this phenotype, suggesting that the expression of ICP0 is crucial for cell cycle arrest. Analysis of global cellular gene expression patterns following infection with d106 and d109 revealed that a relatively small subset of cellular genes were induced as a consequence of ICP0 expression. A number of these genes induced in the presence of ICP0 are classically considered p53-responsive genes, including p21, gadd45, and mdm-2. However, infection with d106 of cells with both alleles of p53 deleted resulted in the same cell cycle arrest phenotype and similar cellular gene expression patterns, suggesting that the expression of ICP0 results in cell cycle arrest potentially via p53-dependent and p53-independent mechanisms. In addition, it was found that the effects of infection with d106 on viral and cellular gene expression were similar to the effects observed following treatment of cells with the histone deacetylase inhibitor trichostatin A.

The polyserine tract of herpes simplex virus ICP4 is required for normal viral gene expression and growth in murine trigeminal ganglia.

ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (DeltaSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. DeltaSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [tk] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a tk mutant, dlsptk. Both DeltaSER and dlsptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.

Herpes simplex virus for gene delivery to neurons.

Vectors derived from herpes simplex virus provide a means of gene delivery to postmitotic neurons. The virus is readily taken up at nerve terminals, passes by rapid retrograde and anterograde transport within neurons, and is selectively transferred across synapses, thus allowing it entry from the periphery into the brain. This virus can enter a state of latency in some neurons, where it exists as an episomal element in the nucleus and is transcriptionally active to a reduced extent. In this state, the virus is apparently benign and can effect stable expression of foreign genes. The large (150 kb) genome of this double-stranded virus has been completely sequenced. Many of its 70 genes can be replaced while still allowing the virus to replicate in at least some cultured cells. Some mutations in the viral genome can compromise the toxicity of the virus and reduce or eliminate its ability to replicate within neurons. Many uses for herpes vectors can be envisioned, including evaluation of neuronal promoter elements and functions of neural proteins in culture and in vivo, as well as therapeutic delivery of genes to modulate nerve function and for gene replacement therapy in vivo.

A second-site revertant of a defective herpes simplex virus ICP4 protein with restored regulatory activities and impaired DNA-binding properties.

A mutant of herpes simplex virus type 1, vi12, encodes a DNA-binding- and transactivation-deficient ICP4 polypeptide. Because of the mutation, the vi12 virus does not grow on Vero cells but must be propagated on cells that express complementing levels of wild-type ICP4 (E5 cells). A pseudorevertant of vi12, designated pri12, was isolated on the basis of the restored ability to replicate on Vero cells. In addition to the original i12 insertion mutation at amino acid 320, the ICP4 molecule expressed from pri12 possesses an alanine to valine substitution at amino acid 342 within the ICP4 gene. The infectivity of pri12 on Vero cells as measured by burst size is elevated by 5 orders of magnitude relative to that observed for vi12, reflecting the restored ability of the mutant ICP4 molecule possessing the alanine to valine substitution to activate transcription and thus support viral replication. Despite the restored regulatory activities of the pri12 ICP4 molecule, the ability of the pseudorevertant ICP4 molecule to form a high-affinity, specific interaction with the consensus binding site was still impaired relative to that of wild-type ICP4. This observation suggests that the in vitro-measured DNA-binding properties of ICP4 may not reflect the functional interactions occurring in vivo that mediate transcriptional activation.

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

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 Km 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.

Analysis of phosphorylation sites of herpes simplex virus type 1 ICP4.

The herpes simplex virus ICP4 protein is required for induction of early and late viral gene transcription as well as for repression of expression of its own gene and several other viral genes. Several electrophoretic forms of ICP4 have been observed, and phosphorylation is thought to contribute to this heterogeneity and possibly to the multiple functions of ICP4. To define the complexity of the site(s) of phosphorylation of ICP4 and to initiate mapping of this site(s), we have performed two-dimensional phosphopeptide mapping of wild-type and mutant forms of ICP4 labeled in infected cells or in vitro. Wild-type ICP4 labeled in infected cells shows a complex pattern of phosphopeptides, and smaller mutant forms of ICP4 show progressively fewer phosphopeptides, arguing that multiple sites on ICP4 are phosphorylated. The serine-rich region of ICP4, residues 175 to 198, was shown to be a site for phosphorylation. Furthermore, the serine-rich region itself or the phosphorylation of this region increases phosphorylation of all phosphopeptides. A mutant ICP4 molecule lacking the serine-rich region showed low levels of phosphorylation by protein kinase A or protein kinase C in vitro. These results suggest that there may be a sequential phosphorylation of ICP4, with phosphorylation of the serine-rich region stimulating phosphorylation of the rest of the molecule. In addition, purified ICP4 showed an associated kinase activity or an autophosphorylation activity with properties different from those of protein kinase A or protein kinase C.

Relationship between TATA-binding protein and herpes simplex virus type 1 ICP4 DNA-binding sites in complex formation and repression of transcription.

The herpes simplex virus (HSV) regulatory protein, infected-cell polypeptide 4 (ICP4), represses the transcription of promoters that have binding sites for ICP4 located near the transcription start site. It also been shown that ICP4 binds such promoter DNA cooperatively with the TATA-binding protein (TBP) and TFIIB to form a tripartite protein-DNA complex (C. Smith, P. Bates, R. Rivera-Gonzales, B. Gu, and N. A. DeLuca, J. Virol. 67:4676-4687, 1993). In this study, we analyzed the effects of position and orientation of the ICP4-binding site relative to the TATA box in the ICP4 promoter on transcriptional repression by ICP4 and on the ability of ICP4 to form tripartite complexes with TBP and TFIIB. The results of theis parallel study provide a strong correlation between tripartite complex formation and repression. Both tripartite-complex formation and transcriptional repression were efficient when the ICP4-binding site was downstream of the TATA box, within a short distance and in proper orientation. In addition, both tripartite-complex formation and repression were partially sensitive to the stereoaxial positioning of the ICP4-binding site relative to the TATA box. As a preliminary characterization of the tripartite complex, circular permutation analysis was performed to assess the distortion of the proximal promoter region in the tripartite complex. As previously reported, both TBP and ICP4 independently could bend DNA and the relative magnitude by which each of these proteins bent DNA in the tripartite complex was preserved. The results of this study suggest that the formation of tripartite complexes on a promoter is part of the mechanism of repression and that simple blocking as a sole result of ICP4 binding is not sufficient for full repression.

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

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.

Development and application of herpes simplex virus vectors for human gene therapy.

Advances in understanding the molecular basis of human disease and the development of recombinant DNA methods is rapidly creating new means of disease diagnosis and treatment. Among the most revolutionary developments are technologies for transfer of therapeutic genes to the human body to treat both inherited and acquired disease. Gene therapy offers considerable promise for ameliorating otherwise intractable diseases such as immunopathological conditions, cancer, heart disease, and various metabolic and neurodegenerative syndromes. To fulfill this promise, more efficient and effective methods of gene delivery and appropriate gene expression must be developed. The lack of such techniques is currently the most significant impediment to the use of genetic therapy. Both viral and nonviral delivery systems are under development for specific gene-therapy applications. Herpes simplex virus (HSV) represents a novel vector system for gene delivery to the nervous system and other tissues. HSV is able to establish latency in nondividing neuronal cells in which genomes persist long-term but do not integrate or alter host-cell metabolism and that carry a promoter system uniquely capable of escaping repression that shuts off the expression of HSV-lytic genes during latency. This review examines efforts to create defective HSV vectors that are safe, noncytotoxic, and applicable to the treatment of cancer and diseases affecting peripheral nerves. Perhaps the most important use of HSV vectors will be for the treatment of neurodegenerative diseases of the brain, but additional studies are required to improve the design of promoters to ensure regulatable or effective levels of therapeutic gene expression.

Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it has been shown previously that ICP0 is present predominantly in the cytoplasms of cells infected with an ICP4 null mutant virus (n12) at high multiplicities of infection and that the level of ICP27, a third viral regulatory protein, plays an important role in determining the intracellular localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer, J. Virol. 68:3027-3040, 1994). To address whether the cytoplasmic localization of ICP0 is a common feature of cells infected with all ICP4 mutant viruses or whether mutant ICP4 polypeptides, together with ICP27, determine the intracellular localization of ICP0, we used double-staining immunofluorescence tests to examine the intracellular staining patterns of ICP0 and ICP4 in cells infected with an extensive series of ICP4 mutant viruses. In these tests, compared with the localization pattern of ICP0 in wild-type virus-infected cells, more ICP0 was detected in the cytoplasms of cells infected with all ICP4 mutants tested at high multiplicities of infection. Each of the mutant forms of ICP4 exhibiting predominantly cytoplasmic staining contains both the nuclear localization signal and the previously mapped ICP27-responsive region (Z. Zhu and P. A. Schaffer, J. Virol. 69:49-59, 1995). No correlation between the intracellular staining patterns of ICP0 and mutant forms of ICP4 was demonstrated, suggesting that mutant ICP4 polypeptides per se are not responsible for retention of ICP0 in the cytoplasm. This observation was confirmed in studies of cells cotransfected with plasmids expressing ICP0 and mutant forms of ICP4, in which the staining pattern of ICP0 was not changed in the presence of mutant ICP4 proteins. Studies of cells infected at low multiplicities with a variety of ICP4 ts mutant viruses at the NPT showed that both ICP0 and ts forms of ICP4 were localized predominantly within the nucleus. These observations are a further indication that the aberrant localization of the ts forms of ICP4 at the NPT is not a direct result of specific mutations in the ICP4 gene. In the final series of tests, the localization of ICP0 in cells infected with a double-mutant virus unable to express either ICP4 or ICP27 was examined. In these tests, ICP0 was detected exclusively in the nuclei of Vero cells but in both the nuclei and the cytoplasms of ICP27-expressing cells infected with the double mutant. These results demonstrate that ICP27, rather than the absence of functional ICP4, is responsible for the cytoplasmic localization of ICP0 in ICP4 mutant virus-infected cells. Taken together, these findings demonstrate that the aberrant localization of ICP0 and certain mutant forms of ICP4 in cells infected with ICP4 mutant viruses is mediated by high levels of ICP27 resulting from the inability of mutant forms of ICP4 to repress the expression of ICP27.