Mapping of functional regions in the amino-terminal portion of the herpes simplex virus ICP27 regulatory protein: Importance of the leucine-rich nuclear export signal and RGG box RNA-binding domain

During its lytic infection, herpes simplex virus type 1 (HSV-1) exerts tight regulatory control over the expression of its genes (for a review of HSV gene regulation, see reference 38). The approximately 80 viral genes are transcribed in the infected-cell nucleus by the host RNA polymerase II and are expressed in three sequential waves. Immediately upon infection, five immediate-early (IE) genes are transcriptionally induced via the action of a virus-encoded virion protein, VP16, working in concert with cellular proteins. Four of the five IE genes encode nuclear proteins (ICP0, ICP4, ICP22, and ICP27) which activate the expression of all later genes. Upon synthesis of the IE proteins, delayed-early (DE) genes are expressed. Many of the DE proteins have enzymatic activities which are involved directly or indirectly in viral DNA replication. Accumulation of these proteins results in the activation of viral DNA replication, which in turn stimulates the expression of the last set of genes, the late (L) genes. These genes predominantly encode viral structural proteins and can be subdivided into leaky-L and true-L classes, depending on whether they are partially or wholly dependent upon viral DNA synthesis for their expression. In addition to sequentially activating its own genes during infection, HSV-1 also uses multiple mechanisms to inhibit the expression of cellular genes. Of the four IE proteins which regulate viral gene expression, ICP4 and ICP27 are essential for growth under all known conditions, whereas ICP0 and ICP22 are required only under certain conditions. ICP4 is the major transcriptional activator protein of HSV-1, being stringently required for DE/L gene transcription. The role of ICP27 is less well characterized, but it appears to carry out multiple functions. First, studies of viral ICP27 mutants have shown that it plays a critical role in stimulating the expression of certain DE/L gene mRNAs and proteins (21, 22, 24, 33, 35, 40, 50). ICP27 mutants express reduced levels of several DE genes encoding essential DNA replication factors (22, 50). This effect appears to be responsible for the reduced levels of viral DNA replication that are observed in ICP27 mutant-infected cells. Second, ICP27 represses the expression of several viral IE and DE genes at late times after infection (21, 33, 40). Third, ICP27 inhibits the production of host mRNAs and proteins during infection (14, 40, 46, 49). The molecular mechanisms by which ICP27 exerts its multiple effects on gene expression are only partially understood. However, many of ICP27's effects are mediated at the posttranscriptional level. Consistent with this, ICP27 is an RNA-binding protein (4, 18, 27, 42), although the biological significance of this activity is presently unclear. It is also not known whether ICP27 binds to specific RNA sequences or structures. ICP27's posttranscriptional effects on gene expression are diverse. It inhibits pre-mRNA splicing during the lytic infection (5, 16), an effect which is at least partially responsible for ICP27's inhibitory effects on host gene expression. ICP27 also modulates the efficiency of pre-mRNA polyadenylation (23) and in so doing may stimulate the expression of certain L genes which contain inherently weak polyadenylation sites (22). It has been proposed that ICP27 functions as a transport factor for the nuclear export of viral L mRNAs, which, due to their lack of introns, may not be able to efficiently access the normal cellular mRNA export pathway (20, 42, 46). Consistent with a role in mRNA export, ICP27 has been found to shuttle continuously between the nucleus and cytoplasm (26, 30, 42, 46). Evidence that ICP27 affects nuclear export pathways in infected cells also comes from studies which show that it promotes the cytoplasmic accumulation of unspliced alpha-globin mRNA in HeLa cells (7, 10). ICP27 also affects mRNA stability, in that it posttranscriptionally stabilizes beta interferon mRNA in infected cells (4, 28). In addition to these seemingly diverse posttranscriptional effects, ICP27 may also regulate transcription during infection, as it appears to stimulate the transcription of at least two viral L genes (19) and inhibit the transcription of certain cellular genes (49). The functional domains of ICP27 have not been fully delineated. Numerous studies, however, indicate that the carboxyl- (C)-terminal half of the protein (from approximately residue 260 to the C terminus at residue 512) is critical (4, 15, 24, 33, 34, 47, 48). Consistent with this, the sequences corresponding to the C-terminal half of ICP27 are conserved in regulatory proteins encoded by all mammalian and avian herpesviruses sequenced to date (39). In contrast, the amino (N)-terminal half of ICP27 also has important functional regions but is not closely conserved in sequence among herpesviruses. In the first 200 amino acid residues of ICP27, at least three functional regions have been described (Fig. ​(Fig.1),1), each of which can function in the context of heterologous proteins. These are (i) a leucine-rich nuclear export signal (NES), similar to that of the human immunodeficiency virus type 1 Rev protein, mapping to residues 5 to 15 (42); (ii) a nuclear localization signal (NLS), mapping to residues 109 to 137 (25); and (iii) an RGG box RNA-binding domain, mapping to residues 137 to 153 (27). The N-terminal half of ICP27 is also the site of multiple posttranslational modifications, including phosphorylation of serine residues (52), methylation of one or more arginine residues on the RGG box (27), and possibly nucleotidylylation (2, 3). FIG. 1. HSV-1 ICP27 N-terminal mutants. (A) The bar at the top represents the ICP27 polypeptide, highlighting known motifs and functional sequences. Below the bar, numbers 1 to 7 refer to sites of engineered XhoI restriction sites (34) which were used to construct ... To date, extensive phenotypic characterization of HSV-1 ICP27 N-terminal mutants is limited, but the phenotype of one mutant, d1-2, has been studied in detail (35). This mutant lacks residues 12 to 63, which comprise a highly acidic region. d1-2 is deficient for growth in Vero cells, exhibiting somewhat reduced levels of viral DNA replication and L gene expression. However, d1-2 is not tightly blocked for expression of several L genes, including the true-L gene encoding glycoprotein C (gC). This phenotype differs from those of several C-terminal ICP27 mutants, which are tightly restricted from expressing gC and several other L genes (33, 34, 45, 46, 48). The deletion in d1-2 impinges on the NES but does not entirely remove it (Fig. ​(Fig.1B).1B). It is therefore unclear to what extent the NES disruption contributes to the phenotype of d1-2. To clarify the role of the NES in ICP27 function, we have engineered a viral mutant, dLeu, which specifically lacks the NES (residues 6 to 19). We have compared its phenotype to those of d1-2 and several other N-terminal ICP27 deletion mutants. These studies have allowed us to genetically define the functional regions in the N-terminal 200 residues of ICP27.