The herpes simplex virus 1 ICP27 is an essential, highly conserved protein involved in various steps of herpes simplex virus 1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. These activities are discussed.
- gene expression
- ribonucleoprotein (RNP)
- herpes simplex virus
- nuclear export
- pre-mRNA splicing
Human HSV-1 (herpes simplex virus type 1) is a nuclear replicating DNA virus. After infection, the 152 kb genome, encoding at least 80 genes, circularizes and is transcribed by the host RNA polymerase II. The viral genes are expressed in a temporarily co-ordinated fashion and can be divided into three classes: α (immediate early), β (delayed early) and γ (late). Transcription of the five α genes is stimulated by the virion-associated VP16 protein and their expression does not require prior protein synthesis. Expression of the β proteins, which are involved in viral DNA replication and metabolism, is regulated by α gene products. The γ gene class, necessary for virion assembly, maturation and egress, can be subdivided into the γ1 genes, whose expression is stimulated by viral DNA synthesis but is not dependent on it, and the γ2 genes, whose expression shows an absolute requirement for viral DNA replication (reviewed in ). HSV-1 has profound effects on the metabolism of infected cells by efficiently repressing host protein synthesis. This is achieved through nonspecific destabilization of mRNA by a virion-associated protein and subsequent suppression of the remaining host protein synthesis by another activity that is dependent on viral gene expression (reviewed in [1,2]).
ICP27 is a 63 kDa immediate-early regulatory phosphoprotein that is essential for HSV-1 lytic replication and is conserved among all mammalian and avian herpesviruses sequenced so far. Early work on ICP27 implicated it both in the regulation of β and γ viral gene expression and in the shut-off of host protein synthesis [3⇓⇓–6]. More specifically, use of viral ICP27 mutants showed that this protein is necessary for efficient DNA replication by promoting the accumulation of β proteins and, at later times, for the transition from β to γ gene expression by activating γ genes while repressing α and β genes [7⇓⇓–10]. Much recent work in several laboratories has demonstrated that, although ICP27 may have a role in transcription , it exerts these effects mainly at various levels of post-transcriptional gene regulation.
Properties of ICP27 and its functional domains
ICP27 binds RNA through an arginine- and glycine-rich motif, the RGG-box (Figure 1) [12⇓–14]. In addition to this region, the N-terminus of the protein has been proposed to contain nuclear export and localization signals that promote shuttling of ICP27 between the nucleus and cytoplasm as explained later [13,15]. The C-terminus, necessary for both transactivation and repression functions , contains three domains with homology to RNA-binding regions of hnRNP (heterogeneous nuclear ribonucleoprotein) K . These KH-like regions (KH1–KH3) are required for γ gene transactivation and for shuttling of ICP27 [17⇓–19], but have not been shown to bind RNA directly. Partially coinciding with KH3 is a putative zinc-finger domain required for ICP27 self-interaction [20,21] and a proposed Sm domain, homologous with the protein–protein interaction motif of certain spliceosomal proteins, that is associated with host shut-off [17⇓–19].
ICP27 inhibits splicing of pre-mRNA (precursor mRNA)
Transfection experiments demonstrated that ICP27 can act either as an activator or as a repressor of expression depending on the target gene . Sandri-Goldin and Mendoza  showed that the regulatory activity of ICP27 depends on the presence of different mRNA processing signals in the target mRNA. The activation function correlates with weak polyadenylation sites that are activated by ICP27 through an unknown mechanism , whereas the repressor function correlates with the presence of introns . Subsequent investigations showed that ICP27 is clearly involved in the inhibition of pre-mRNA splicing at early stages of HSV-1 infection [5,24] and that it can inhibit splicing in the absence of other viral factors . Because, in contrast with the cellular genome, all but four HSV-1 genes are intronless, negative regulation of splicing specifically contributes to shut-off of host protein synthesis, a process in which genetic studies had previously implicated ICP27 . The splicing reaction is inhibited at an early stage before the first catalytic step [25⇓–27], but how this occurs is not yet understood, although interactions of ICP27 with various cellular proteins associated with splicing have been demonstrated. During HSV-1 infection, snRNPs (small nuclear ribonucleoproteins) that are directly involved in splicing  are redistributed in the nucleus ; ICP27 is responsible for this effect and the process requires C-terminal regions of the protein [30,31]. Although ICP27 co-localizes with the redistributed snRNPs and alters the phosphorylation of at least one of them [30⇓–32], there is evidence that these effects by themselves are insufficient to inhibit splicing [31,32]. ICP27 interacts with and redistributes a number of splicing regulatory factors such as SR proteins, which participate in spliceosome assembly [31,33], and p32, an inhibitor of the SF2 SR protein [34,35]; however, the effects of these interactions on splicing have not been directly investigated. The association of ICP27 with another regulator of SR proteins, SRPK1 (SR protein kinase 1) , was reported to direct this factor from the cytoplasm to the nucleus, resulting in an inhibitory hypophosphorylation of SR proteins and stalling of spliceosome assembly . In a separate study , ICP27, but not mutant forms of the protein deficient in host shut-off or splicing inhibition, was found to bind to and co-localize with SAP145 (spliceosome-associated protein 145), another essential splicing complex assembly factor . In summary, it is apparent that ICP27-mediated inhibition of splicing plays an important part in HSV-1-induced host shut off, but its mechanism and the functions of the different interactions of ICP27 with pre-mRNA processing factors remain to be established.
ICP27 shuttles between the nucleus and cytoplasm and exports HSV-1 mRNA
In HSV-1-infected cells, intron-containing transcripts were found to be preferentially retained in the nucleus at the sites of redistributed snRNPs in an ICP27-dependent manner, first suggesting an involvement of this protein in the regulation of nucleocytoplasmic RNA transport . It has since been demonstrated that ICP27 shuttles between the nucleus and cytoplasm [13,18,39⇓–41] and that it binds specifically to intronless mRNAs in both cellular compartments , indicative of a direct role in the nuclear export of viral RNA. In keeping with this, the exit of ICP27 from the nucleus has been reported to be stimulated by some viral γ gene mRNAs [17,18]. Shuttling of ICP27 depends both on the RGG-box to bind RNA and reportedly on the presence of a leucine-rich region, homologous with the NES (nuclear export signal) of the HIV-1 Rev protein (Figure 1) [13,42]. Rev mediates the export of HIV-1 mRNA by binding the cellular nuclear transport receptor CRM1 through its NES, an interaction that is inhibited by the compound LMB (leptomycin B) (reviewed in ). Surprisingly, despite its dependence on the NES, ICP27 accumulates in the cytoplasm in the presence of LMB [41,44], indicating that a CRM1-independent export pathway is being used. In a previous report to the contrary , LMB sensitivity was inferred from a study in which nuclear re-import of protein was allowed to occur, complicating the interpretation of the results since the steady-state distribution of ICP27 is predominately nuclear .
Cellular mRNA appears to be exported through a pathway that is mediated by the nuclear export receptor TAP in conjunction with an RNA-binding protein REF, a constituent of the exon junction complex . ICP27 interacts directly with REF and, in infected cells, relocates it from sites of cellular splicing to sites of viral transcription . Furthermore, overexpression of REF in HSV-1-infected cells stimulates the export of several viral mRNAs ; in a study where viral intronless mRNAs were injected into Xenopus oocyte nuclei, co-injection of ICP27, but not of mutant protein incapable of binding REF, stimulated viral mRNA export . In contrast with its activity in cellular mRNA export, REF does not bind directly to HSV-1 transcripts , probably because they lack introns, suggesting a model where ICP27 binds the viral mRNA and, through its interaction with REF, acts as an adaptor to export the transcripts through the TAP pathway. However, several important observations remain unexplained with this model. (i) Deletion of either the RGG-box or the REF-binding site from ICP27 has only moderate effects on viral gene expression , (ii) export of a number of viral transcripts appears to be LMB-sensitive and thus CRM1-dependent  and (iii) export of transcripts of some genes whose overall expression is dependent on ICP27 can occur in its absence . It is therefore probable that the TAP/REF pathway is not the sole system for exporting viral transcripts and it is possible that ICP27 contains additional domains involved in nuclear export. It is noteworthy, in this respect, that ICP27 has homology to the KNS domain of hnRNP K that mediates both nuclear import and export by an as-yet unknown mechanism . Furthermore, some viral transcripts may depend entirely on host export pathways or, alternatively, there may exist viral proteins other than ICP27 with activities in nuclear export of RNA. For instance, a component of the HSV-1 tegument, VP13/14, has recently been shown to shuttle between the nucleus and the cytoplasm and contains a leucine-rich NES-like element, but no information yet exists concerning a role for this factor in gene expression .
ICP27 homologues in other herpesviruses
Some homologues of ICP27 in other herpesviruses, such as the human cytomegalovirus UL69 protein, the Epstein–Barr virus Sm protein and the ORF57 gene products of KSHV (Kaposi's sarcoma-associated herpesvirus) and herpesvirus saimiri have been shown to be capable of nucleocytoplasmic shuttling [48⇓⇓–51]. Recently, KSHV ORF57 was found to interact with REF and to export RNAs that are poor splicing substrates; thus it appears to play an analogous role to ICP27 in this respect . Epstein–Barr virus Sm has been reported to inhibit splicing  and to be involved in mRNA export; however, there are conflicting reports concerning the pathway employed for this [54,55].
ICP27 has several activities in gene expression, two of which, splicing and RNA export, have been discussed at length in this review. It is not yet clear how the various functions of ICP27 contribute to the tight temporal regulation of HSV-1 gene expression. An important issue in this regard is the question of which proteins are associated with ICP27, which mRNAs are bound to it and how these interactions change during the course of infection. ICP27 is an essential gene but it is not apparent which of its individual functions are absolutely required. In this respect, it is interesting that some late gene products that do not require ICP27 for export still depend on ICP27 for their expression. Studies are now underway to investigate further the cytoplasmic activities of ICP27, such as in translation.
RNA Structure and Function: Joint Biochemical Society/Royal Society of Chemistry Focused Meeting held at the Michael Swann Building, University of Edinburgh, U.K., 4–6 December 2004. Organized and Edited by S.V. Graham (Glasgow, U.K.) and D.M.J. Lilley (Dundee, U.K.). Sponsored by BBSRC (Biotechnology and Biological Sciences Research Council), Glen Research, Promega UK Ltd, VH Bio Ltd, Stratagene, New England Biolabs (UK) Ltd, MWG Biotech UK Ltd, Ambion Europe Ltd and Link Technologies Ltd.
Abbreviations: hnRNP, heterogeneous nuclear RNP; HSV-1, herpes simplex virus type 1; LMB, leptomycin B; NES, nuclear export signal; snRNP, small nuclear RNP
- © 2005 The Biochemical Society