Rozanne M. Sandri-Goldin

Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression

The herpes simplex virus (HSV-1) immediate early protein ICP27 is a regulatory protein which is essential for virus replication. The phenotype of temperature-sensitive and deletion mutants in ICP27 includes overexpression of some immediate early and early gene products and greatly reduced levels of late gene products. To determine whether regulation by ICP27 occurs primarily at the transcriptional level, we have studied the expression of two immediate early products (ICP4 and ICP27) and two late gene products (glycoprotein B and glycoprotein C) at the level of transcription initiation, accumulation of steady state mRNA, and protein synthesis in an ICP27 temperature-sensitive mutant tsLG4, compared to wild-type HSV-1. At the nonpermissive temperature in tsLG4-infected cells, the two immediate early gene products ICP4 and ICP27 were overexpressed both at the mRNA and protein level although synthesis of these transcripts as measured by nuclear runoff assays was reduced relative to the wild-type HSV-1 infections. The transcription of late gene products glycoprotein B (gB) and glycoprotein C (gC) was lower in runoff assays from tsLG4 infections suggesting that the reduction in the level of late products occurred at the transcriptional level. However, temperature shift experiments in which tsLG4-infected cells were shifted to the nonpermissive temperature at various times after infection showed that the synthesis of late transcripts was not altered 2 hr after the shift whereas both the accumulation of leaky late and late mRNA and the incorporation of [35S]methionine into newly synthesized gB and gC was reduced by 2 hr after the shift to nonpermissive temperature. Therefore, while the synthesis of new transcripts continued, the accumulation of late mRNAs and their translation into protein was reduced when ICP27 was defective, whereas, the converse was found for immediate early products. That is, the synthesis of new transcripts was reduced yet mRNA and protein accumulated to high levels. These results suggest that ICP27 acts at least in part post-transcriptionally to regulate the expression of immediate early and late gene products.

Global Analysis of Herpes Simplex Virus Type 1 Transcription Using an Oligonucleotide-Based DNA Microarray

ABSTRACT More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early α27 (UL54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the α27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.

ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation

Infection with some viruses can alter cellular mRNA processing to favor viral gene expression. We present evidence that herpes simplex virus 1 (HSV‐1) protein ICP27, which contributes to host shut‐off by inhibiting pre‐mRNA splicing, interacts with essential splicing factors termed SR proteins and affects their phosphorylation. During HSV‐1 infection, phosphorylation of several SR proteins was reduced and this correlated with a subnuclear redistribution. Exogenous SR proteins restored splicing in ICP27‐inhibited nuclear extracts and SR proteins isolated from HSV‐1‐infected cells activated splicing in uninfected S100 extracts, indicating that inhibition occurs by a reversible mechanism. Spliceosome assembly was blocked at the pre‐spliceosomal complex A stage. Furthermore, we show that ICP27 interacts with SRPK1 and relocalizes it to the nucleus; moreover, SRPK1 activity was altered in the presence of ICP27 in vitro. We propose that ICP27 modifies SRPK1 activity resulting in hypophosphorylation of SR proteins impairing their ability to function in spliceosome assembly.

ICP27 Interacts with the RNA Export Factor Aly/REF To Direct Herpes Simplex Virus Type 1 Intronless mRNAs to the TAP Export Pathway

ABSTRACT Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV-1-infected cells but not in Xenopus oocytes, where ICP27 interacts with the export factor Aly/REF to access the TAP export pathway. Here, we show that ICP27 interacts with Aly/REF in HSV-1-infected mammalian cells and that Aly/REF stimulates export of viral intronless RNAs but does not cross-link to these RNAs. During infection, Aly/REF was no longer associated with splicing factor SC35 but moved into structures that colocalized with ICP27, suggesting that ICP27 recruits Aly/REF from spliceosomes to viral intronless RNAs. Further, ICP27 was found to interact in vivo with TAP but not with CRM1. In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

The many roles of the regulatory protein ICP27 during herpes simplex virus infection.

Herpes simplex virus type 1 (HSV-1) protein ICP27 is a multifunctional regulator of gene expression that assumes different roles during the course of infection. Early in infection, ICP27 mediates the inhibition of cellular splicing, whereas, later it helps to recruit cellular RNA polymerase II (RNAP II) to viral replication sites and to facilitate viral RNA export. ICP27 has also been shown to stimulate translation of viral transcripts. ICP27 performs its activities by interacting with RNA and with an assortment of proteins. ICP27 binds viral RNAs in its role as an export adaptor. An ever increasing number of cellular proteins have been shown to interact with ICP27, including splicing factors, export proteins and RNAP II. A number of protein motifs within ICP27 have been predicted based upon sequence comparisons; however, detailed structural information is not yet available. Although much has been learned about the mechanisms by which ICP27 performs its roles, relatively little is known about how its activities are regulated. The roles and activities of ICP27 are the subject of this review.

ICP27 Recruits Aly/REF but Not TAP/NXF1 to Herpes Simplex Virus Type 1 Transcription Sites although TAP/NXF1 Is Required for ICP27 Export

ABSTRACT Herpes simplex virus type 1 (HSV-1) protein ICP27 interacts with the cellular export adaptor protein Aly/REF, which is part of the exon junction complex implicated in cellular mRNA export. We previously reported that Aly/REF was no longer associated with splicing factor SC35 sites during infection but instead colocalized with ICP27 in distinct structures. Here we show that these structures colocalize with ICP4 and are sites of HSV-1 transcription. ICP27 mutants with lesions in the region required for the interaction with Aly/REF failed to recruit Aly/REF to viral transcription sites; however, ICP27 export to the cytoplasm was unimpaired, indicating that the interaction of ICP27 with Aly/REF is not required for ICP27 shuttling. ICP27 has also been shown to interact with the cellular mRNA export receptor TAP/NXF1. We report that ICP27 interacts directly with TAP/NXF1 and does not require Aly/REF to bridge the interaction. The C terminus of ICP27 is required; however, the N-terminal leucine-rich region also contributes to the interaction of ICP27 with TAP/NXF1. In contrast to the results found for Aly/REF, mutants that failed to interact with TAP/NXF1 were not exported to the cytoplasm, and TAP/NXF1 was not recruited to sites of HSV-1 transcription. Therefore, the interaction of ICP27 with TAP/NXF1 occurs after ICP27 leaves viral transcription sites. We conclude that ICP27 and the viral RNAs to which it binds are exported via the TAP/NXF1 export receptor.

ICP27 Interacts with the C-Terminal Domain of RNA Polymerase II and Facilitates Its Recruitment to Herpes Simplex Virus 1 Transcription Sites, Where It Undergoes Proteasomal Degradation during Infection

ABSTRACT Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3′ processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.

The many roles of the highly interactive HSV protein ICP27, a key regulator of infection

Human herpes viruses cause an array of illnesses ranging from cancers for Epstein?Barr virus and Kaposi?s sarcoma-associated herpes virus, to painful skin lesions, and more rarely, keratitis and encephalitis for HSV. All herpes viruses encode a multifunctional protein, typified by HSV ICP27, which plays essential roles in viral infection. ICP27 functions in all stages of mRNA biogenesis from transcription, RNA processing and export through to translation. ICP27 has also been implicated in nuclear protein quality control, cell cycle control, activation of stress signaling pathways and prevention of apoptosis. ICP27 interacts with many proteins and it binds RNA. This article focuses on how ICP27 performs its many roles and highlights similarities with its homologs, which could be targets for antiviral intervention.

Viral Regulation of mRNA Export

Recent advances have led to an understanding of how eukaryotic mRNAs are exported from the nucleus to the cytoplasm. This process involves an elaborate machinery that is conserved from yeasts to humans and is coupled to upstream events in RNA metabolism. Eukaryotic pre-mRNAs are processed after synthesis in the nucleus by capping at the 5′ end, cleavage and polyadenylation to form the 3′ end, and splicing to remove intervening sequences. Following processing, mRNAs must be exported through the nuclear pore complex (NPC) to the cytoplasm for translation, which requires recognition by export factors to direct the mRNAs to nuclear export receptors for translocation through the NPC (17, 81, 108). A large body of evidence shows that TAP/NXF1, in conjunction with its heterodimeric partner, p15/NXT, is the nuclear export receptor for mRNAs in metazoans (5, 27, 33, 45, 48, 107) (Table ​(Table1).1). The yeast homologue of TAP/NXF1, termed Mex67p, has been shown to function as the mRNA export receptor in yeasts (44, 88, 91, 101). TAP/NXF1 and Mex67p have been shown to shuttle between the nucleus and cytoplasm, cross-link to poly(A)+ RNA, localize at the nuclear pores, and interact directly with nucleoporins (5, 37, 45, 88, 91). Further, overexpression of TAP/NXF1 in Xenopus oocytes or mammalian cells stimulated mRNA export (5, 37), and inactivation of TAP/NXF1 in Caenorhabditis elegans (103) and Drosophila (38) by RNA interference blocked nuclear export of poly(A)+ RNA, indicating a direct role in mRNA export.