Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins.

The herpes simplex virus mutants KOS1.1 ts756 and HFEM tsLB2 express temperature-sensitive ICP4 proteins that are not localized properly to the cell nucleus at the nonpermissive temperature. In these infected cells at the nonpermissive temperature, nuclear localization of at least two other viral proteins, ICP0 and ICP8, is impaired. Replacement of the mutated sequences in the ICP4 gene of tsLB2 restored proper nuclear localization of all of the proteins. The ICP0 and ICP8 proteins expressed in cells transfected with their individual genes were localized to the cell nucleus. Therefore, in infected cells, the mutant ICP4 gene product appears to be the primary defect which leads to the block in nuclear localization of the other proteins. One viral protein, ICP27, was not inhibited for nuclear localization in these cells. These data indicate that there are at least two pathways for nuclear localization of HSV proteins, one of which is inhibited by the mutant ICP4 protein. The mutant ICP4 protein may define a probe for one of the pathways of nuclear localization of proteins.     

A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid.

The major DNA-binding protein, ICP8, encoded by herpes simplex virus is localized to the infected cell nucleus where it plays a role in viral DNA replication and control of viral gene expression. To identify the parts of the ICP8 protein that are important for its localization and functions, we have developed a system to test the ability of recombinant plasmids to express functional ICP8. A recombinant plasmid containing the wild-type ICP8 gene was transfected into cells. The cells were later infected with a temperature-sensitive ICP8 mutant virus at the nonpermissive temperature. Sufficient wild-type ICP8 was expressed from the transfected plasmid to complement the replication of the mutant virus. This provides a genetic system to test the properties of ICP8 expressed from mutagenized plasmids without the establishment of a stable cell line or the reintroduction of the ICP8 gene into the herpes simplex virus genome.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein.

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.     

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.     

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.     

Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus

To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.     

The two functions of herpes simplex virus 1 ICP0, inhibition of silencing by the CoREST/REST/HDAC complex and degradation of PML, are executed in tandem

ICP0, an α (immediate-early) protein of herpes simplex virus 1, performs at least two key functions. It blocks inhibition of viral-gene expression by interferon, a function dependent on the degradation of the ND10 components PML and SP100 by the ubiquitin ligase expressed by the RING finger (RF), and it blocks silencing of viral DNA mediated by the HDAC1/2-CoREST-REST complex. In the latter case, a mutant CoREST lacking the HDAC1 binding site compensates totally or in part for the absence of ICP0 in a cell-type-dependent manner. Here, we compare the phenotypes of an ICP0 mutant containing disabling amino acid substitutions in the RF with those of a mutant with substitutions in the CoREST binding site (R8507). We report the following: (i) the onset of replication of both mutants was delayed, but the RF mutant yields did not reach wild-type virus levels even as late as 48 h after infection, and (ii) in infected cells, PML is rapidly degraded by wild-type virus, with some delay by the R8507 mutant, and is spared by the RF mutant. The translocation of ICP0 to the cytoplasm is impaired in cells infected with the RF mutant or delayed in cells infected with the R8507 mutant. Finally, in contrast to wild-type viruses, both mutants are inhibited by alpha or gamma interferon. The results indicate that both sets of events, the degradation of PML and the blocking of silencing, are interdependent and in large measure dependent on events in the ND10 nuclear bodies.     

Transformation by v-myb correlates with trans-activation of gene expression.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.     

Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus.

We examined the kinetics and the nature of the association of two herpes simplex virus proteins, the major DNA-binding protein (ICP8) and the major capsid protein (ICP5), with the nuclei of infected cells. We defined a series of stages in the association of the ICP8 protein with the cell nucleus. (i) Immediately after synthesis, the protein was found in the cytoplasmic fraction but associated rapidly with the crude nuclear fraction. (ii) The initial association of ICP8 with the crude nuclear fraction was detergent sensitive but DNase resistant, and, thus, the protein was either bound to structures attached to the outside of the nucleus and had not penetrated the nuclear envelope or was loosely bound in the nucleus, (iii) At intermediate times, a low level of an intermediate form was observed in which the association of ICP8 with the nuclear fraction was resistant to both detergent and DNase treatment. The protein may be bound to the nuclear matrix at this stage. Inhibition of viral DNA synthesis caused the DNA-binding protein to accumulate in this form. (iv) At late times during the chase period, the association of ICP8 with the cell nucleus was resistant to detergent treatment but sensitive to DNase treatment. our results argue that at this stage ICP8 was bound to viral DNA. Thus, nuclear association of the DNA-binding protein did not require viral DNA replication. More important is the observation that there is a series of stages in the nuclear association of this protein, and, thus, there may be a succession of binding sites for this protein in the cell during its movement to its final site of action in the nucleus. The major capsid protein showed some similar stages of association with the cell nucleus but the initial association with the nucleus followed a lag period. Its early association with the crude nuclear fraction was also detergent sensitive but was resistant to detergent treatment at later times. Its association with the cell nucleus was almost completely resistant to DNase treatment at all times. Inhibition of viral DNA replication blocked the nuclear transport of this protein. Thus, these two viral proteins share some stages in nuclear transport, although their requirements for nuclear association are different.     

A pre-immediate-early role for tegument ICP0 in the proteasome-dependent entry of herpes simplex virus

Herpes simplex virus (HSV) entry requires host cell 26S proteasomal degradation activity at a postpenetration step. When expressed in the infected cell, the HSV immediate-early protein ICP0 has E3 ubiquitin ligase activity and interacts with the proteasome. The cell is first exposed to ICP0 during viral entry, since ICP0 is a component of the inner tegument layer of the virion. The function of tegument ICP0 is unknown. Deletion of ICP0 or mutations in the N-terminal RING finger domain of ICP0 results in the absence of ICP0 from the tegument. We show here that these mutations negatively influenced the targeting of incoming capsids to the nucleus. Inhibitors of the chymotrypsin-like activity of the proteasome the blocked entry of virions containing tegument ICP0, including ICP0 mutants that are defective in USP7 binding. However, ICP0-deficient virions were not blocked by proteasomal inhibitors and entered cells via a proteasome-independent mechanism. ICP0 appeared to play a postpenetration role in cells that supported either endocytosis or nonendosomal entry pathways for HSV. The results suggest that ICP0 mutant virions are defective upstream of viral gene expression at a pre-immediate-early step in infection. We propose that proteasome-mediated degradation of a virion or host protein is regulated by ICP0 to allow efficient delivery of entering HSV capsids to the nuclear periphery.     

Oligomerization of ICP4 and rearrangement of heat shock proteins may be important for herpes simplex virus type 1 prereplicative site formation

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.     

Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins.

Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Potential role for herpes simplex virus ICP8 DNA replication protein in stimulation of late gene expression.

We have identified a trans-dominant mutant form of the herpes simplex virus (HSV) DNA-binding protein ICP8 which inhibits viral replication. When expressed by the V2.6 cell line, the mutant gene product inhibited wild-type HSV production by 50- to 150-fold when the multiplicity of infection was less than 5. Production of HSV types 1 and 2 but not production of pseudorabies virus was inhibited in V2.6 cells. The inhibitory effect was not due solely to the high levels of expression, because the levels of expression were comparable to those in the permissive wild-type ICP8-expressing S-2 cell line. Experiments designed to define the block in viral production in V2.6 cells demonstrated (i) that viral alpha and beta gene expression was comparable in the different cell lines, (ii) that viral DNA replication proceeded but was reduced to approximately 20% of the control cell level, and (iii) that late gene expression was similar to that in cells in which viral DNA replication was completely blocked. Genetic experiments indicated that the mutant gene product inhibits normal functions of ICP8. Thus, ICP8 may play distinct roles in replication of viral DNA and in stimulation of late gene expression. The dual roles of ICP8 in these two processes could provide a mechanism for controlling the transition from viral DNA synthesis to late gene expression during the viral growth cycle.     

Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis.

Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.     

Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0.

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.