The herpes simplex virus ICP27 protein: a multifunctional post-transcriptional regulator of gene expression.

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.

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway.

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.

Herpes simplex virus IE63 (ICP27) protein interacts with spliceosome-associated protein 145 and inhibits splicing prior to the first catalytic step.

The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection.

Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication.

Wild-type (wt) herpes simplex virus type 1 (HSV-1) suppresses cell death. We investigated the apoptotic pathways triggered during infection with mutant viruses tsk and 27lacZ (which lack functional ICP4 and ICP27 viral proteins, respectively) and examined the mechanisms used by wt HSV-1 to protect against programmed cell death induced by the DNA-damaging compound cisplatin. In our studies, we used BHK and HeLa cells, with similar results. We suggest that a decrease in the levels of Bcl-2 protein is a key event during apoptosis induced by the mutant viruses and that Bcl-2 levels are targeted by (i) a decrease of bcl-2 RNA, (ii) caspase-related proteolysis, and (iii) p38 mitogen-activated protein kinase (p38MAPK)-dependent destabilization of Bcl-2 protein. We show that wt HSV-1, but not the mutant viruses, maintains bcl-2 RNA and protein levels during infection and protects from the cisplatin-induced decrease in bcl-2 RNA; our data suggest that both ICP27 and ICP4 are required for this function. Additionally, wt HSV-1 evades but does not actively block activation of caspases. Although wt HSV-1 induces p38MAPK activation during infection, it prevents p38MAPK-dependent destabilization of Bcl-2 and exploits p38MAPK stimulation to enhance transcription of specific viral gene promoters to increase viral yields.

Interaction between herpes simplex virus type 1 IE63 protein and cellular protein p32.

The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991-28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels.

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

The multifunctional herpes simplex virus IE63 protein interacts with heterogeneous ribonucleoprotein K and with casein kinase 2.

Herpes simplex virus type 1 (HSV-1), the prototype alpha-herpesvirus, causes several prominent diseases. The HSV-1 immediate early (IE) protein IE63 (ICP27) is the only regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far. IE63 is a multifunctional protein affecting transcriptional and post-transcriptional processes, and it can shuttle from the nucleus to the cytoplasm. To identify interacting cellular proteins, a HeLa cDNA library was screened in the yeast two-hybrid system using IE63 as bait. Several interacting proteins were identified including heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional protein like IE63, and the beta subunit of casein kinase 2 (CK2), a protein kinase, and interacting regions were mapped. Confirmation of interactions was provided by fusion protein binding assays, co-immunoprecipitation from infected cells, and CK2 activity assays. hnRNP K co-immunoprecipitated from infected cells with anti-IE63 serum was a more rapidly migrating subfraction than hnRNP K immunoprecipitated by anti-hnRNP K serum. Using anti-IE63 serum, both IE63 and hnRNP K were phosphorylated in vitro by CK2, while in immunoprecipitates using anti-hnRNP K serum, IE63 but not hnRNP K was phosphorylated by CK2. These data provide important new insights into how this key viral regulatory protein exerts its functions.

In Vitro Systems to Analyze HSV Transcript Processing.

During lytic virus replication, herpes simplex virus (HSV) exhibits a closely regulated pattern of viral gene expression and of DNA replication, resulting in virion production (1). Broadly, HSV genes can be divided into immediate early, early, and late categories based on the kinetics of their expression. The five immediate early genes are expressed in the absence of prior viral protein synthesis although their expression is stimulated by a viral tegument protein. Two immediate early proteins are essential for virus replication in vitro and act at the transcriptional (IE 175) and posttranscriptional (IE63) levels to regulate early and late gene expression. Throughout infection, mRNA is synthesized using cellular RNA poly-merase II, which is modified by the action of an immediate early protein (2).

Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm.

Herpes simplex virus type 1 (HSV-1) immediate early protein IE63, an essential nuclear protein, is pleiotropic in function and, at the post-transcriptional level, inhibits RNA splicing, interacts with cellular splicing small nuclear ribonucleoprotein particles (snRNPs), binds RNA and prevents the nucleocytoplasmic transport of intron-containing mRNAs. Here it is reported that IE63 is a nucleocytoplasmic shuttle protein able to travel from snRNP- and RNA-rich nuclear foci to the cytoplasm, where it accumulates during actinomycin D treatment. This newly identified property suggests that IE63 facilitates nuclear export of HSV-1 transcripts, in addition to retaining intron-containing transcripts in the nucleus. The mechanism by which IE63 controls RNA export has yet to be defined.

Traumatic abdominal aortic pseudoaneurysm causing biliary obstruction: a case report and review of the literature.

A traumatic pseudoaneurysm of the suprarenal abdominal aorta was diagnosed in a 58-year-old man 32 years after he received a gunshot wound to the abdomen. Epigastric pain and obstructive jaundice were the presenting symptoms. Repair was performed by intraluminal polytetrafluoroethylene patch aortoplasty with resolution of the biliary obstruction. The literature on traumatic abdominal aortic pseudoaneurysm is reviewed and reveals that this report is the first to describe biliary obstruction caused by such a lesion.

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

A cellular 65-kDa protein recognizes the negative regulatory element of human papillomavirus late mRNA.

Papillomavirus late gene expression is tightly linked to the differentiation state of the host cell. Levels of late mRNAs are only in part controlled by regulation of the late promoter, other posttranscriptional mechanisms exist that reduce the amount of late mRNA in undifferentiated cells. Previously we described a negative regulatory element (NRE) located upstream of the human papillomavirus type 16 late poly(A) site. We have delineated the NRE to a 79-nt region in which a G+U-rich region was the major determinant of NRE activity. UV-crosslinking assays identified a prominent nuclear protein of 65 kDa as the only factor in close contact with the NRE, and a complex of at least five proteins, including the 65-kDa protein, was enriched on NRE-RNA. Binding of the 65-kDa protein was depleted by preincubation with poly(U) Sepharose in high salt, a property characteristic of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF65 and bacterially expressed U2AF65 exhibited NRE binding. The 65-kDa protein bound to the G+U-rich NRE 3' half which shows homology to the B2P2 sequence a known U2AF65 binding site in the alpha-tropomyosin gene, and the G+U-rich element can be replaced by B2P2 in the binding assay. Treatment of cells with phorbol 12-myristate 13-acetate reduced binding of the 65-kDa protein, induced NRE binding of a cytoplasmic protein, and relieved the NRE block on reporter gene expression.

Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site.

Two alternative forms of IgM heavy-chain mRNA are produced from a common precursor mRNA as a result of competition between cleavage/poly(A) addition at the upstream (secretory) poly(A) site and cleavage/poly(A) addition at the downstream (membrane) poly(A) site coupled with splicing. The efficiency of cleavage at the secretory poly(A) site is thought to play a crucial role in this alternative processing. We therefore examined RNA binding factors recognizing the secretory poly(A) site, in the absence of the splicing option, to look for transacting factors that may play a role in cleavage/polyadenylation efficiency at this site. Purified primary B cells produce the secretory form of mu mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of mu mRNA when their antigen receptors are ligated by anti-mu antibodies. We compared RNA binding factors in nuclear extracts from cells produced by these different stimulatory conditions and show that induction of the secretory form of mu mRNA by LPS correlates with the induction of a 28-32-kDa secretory poly(A) site-specific polypeptide which is also present in the plasmacytoma cell line J558L. Visualization of the 28-32-kDa polypeptide in UV cross-linking assays depends on a GU-rich element downstream of the secretory poly(A) site. We show that this GU-rich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We also examined nuclear extracts from B cells doubly stimulated with LPS and anti-mu antibodies in which expression of the secretory form of mu mRNA is selectively inhibited. This inhibition may be due to a down-regulation of polyadenylation at the secretory poly(A) site or an up-regulation of the competitive splicing process. This form of stimulation does not lead to the disappearance of the 28-32-kDa polypeptide, but to an enhanced binding of a 50-55-kDa factor which binds both the secretory and membrane poly(A) site. We report the first detection of changes in RNA binding factors taking place at the secretory poly(A) site which correlate with the expression of different forms of mu mRNA produced by primary B cells under different stimulation conditions.

Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts.

Using in situ hybridization labelling methods, we have determined that the herpes simplex virus type 1 immediate-early protein IE63 (ICP27) affects the cellular localization of virus transcripts. Intronless transcripts from the IE63, UL38, and UL44 genes are rapidly exported to and accumulate in the cytoplasm throughout infection, in either the presence or absence of IE63 expression. The intron-containing transcripts from the IE110 and UL15 genes, while initially cytoplasmic, are increasingly retained in the nucleus in distinct clumps as infection proceeds, and the clumps colocalize with the redistributed small nuclear ribonucleoprotein particles. Infections with the IE63 mutant virus 27-lacZ demonstrated that in the absence of IE63 expression, nuclear retention of intron-containing transcripts was lost. The nuclear retention of UL15 transcripts, which demonstrated both nuclear and cytoplasmic label, was not as pronounced as that of the IE110 transcripts, and we propose that this is due to the late expression of UL15. Infections with the mutant virus 110C1, in which both introns of IE110 have been precisely removed (R.D. Everett, J. Gen. Virol. 72:651-659, 1991), demonstrated IE110 transcripts in both the nucleus and the cytoplasm; thus, exon definition sequences which regulate viral RNA transport are present in the IE110 transcript. By in situ hybridization a stable population of polyadenylated RNAs was found to accumulate in the nucleus in spots, most of which were separate from the small nuclear ribonucleoprotein particle clumps. The IE63 protein has an involvement, either direct or indirect, in the regulation of nucleocytoplasmic transport of viral transcripts, a function which contrasts with the recently proposed role of herpes simplex virus type 1 Us11 in promoting the nuclear export of partially spliced or unspliced transcripts (J.-J. Diaz, M. Duc Dodon, N. Schaerer-Uthurraly, D. Simonin, K. Kindbeiter, L. Gazzolo, and J.-J. Madjar, Nature [London] 379:273-277, 1996), the significance of which is discussed.

Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly.

The herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3'-processing and splicing. Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity. Here, IE63 protein expressed in E. coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs. Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly. HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect. RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns.

Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch.

The essential herpes simplex virus type 1 (HSV-1) immediate-early IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3' processing. We have investigated the role of IE63 in the regulation of viral mRNA 3' processing and of late gene expression. Our in vitro 3' processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3' processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3' processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were recognized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3' processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components on the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre-mRNA 3' processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis.

Bringing core biopsy into a surgical practice.

Minimally invasive diagnostic techniques in evaluating patients with breast disease have been increasingly utilized and accepted by physicians and patients over recent years. The incorporation of stereotactic core needle biopsy and ultrasound-guided core needle biopsy into the office practice of evaluating patients with breast disease by our surgical faculty has been met favorably. These procedures are readily learned by surgeons. The judicious use of these procedures is evidenced by the malignancy rate of core biopsies of 16 per cent, identical to the historical rate for needle localization assisted excisional biopsy at our institution. Core breast biopsy expedites definitive diagnosis and optimizes patient convenience. Reimbursement is highly variable, and active physician participation in negotiating with payors to insure that costs are met is essential.

Intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase subunits during infection of cultured cells.

We have analysed the intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase during infection of cultured cells by indirect immunofluorescence using polyclonal and monoclonal antibodies specific for the R1 and R2 subunits. Three different viruses were used to infect cells, wild-type strain 17+ and two temperature-sensitive mutants, ts 1222, which produces R1 only, and ts 1207, which expresses a normal R2 and an altered R1 that fails to interact with R2 at the nonpermissive temperature because of an amino acid substitution in R1. R1 was detected 2 hr postinfection with all three viruses and remained evenly distributed throughout the cytoplasm. R2 was not observed until 4 hr postinfection and, in contrast to the even distribution of R1, was localised in discrete cytoplasmic foci close to the nucleus. In double-labelling experiments both R1 and R2 were found in these foci where they presumably associate to form the active enzyme. As expected R2 was not detectable in cells infected with ts 1222. In ts 1207-infected cells it formed wild-type-like foci, indicating that interaction with R1 is not required for R2 focus formation. R1 was present in a twofold excess over R2 in wild-type-infected cells. We suggest that the uncomplexed R1 could perform a role associated with the protein kinase present in the N-terminal domain.

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.

Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2.

The herpes simplex virus (HSV) gene RL1 encodes the protein ICP34.5, which is a specific neurovirulence factor. Null mutants in RL1 fail to replicate in the central nervous system of mice and are therefore totally non-neurovirulent. Additionally, they fail to replicate in neurons of the peripheral nervous system, although they are capable of establishing and reactivating from a latent infection. As the precise function of ICP34.5 in HSV-neuronal interactions is unknown, we have studied the role of ICP34.5 in vitro by examining in detail the phenotypes of RL1-negative viruses in two defined tissue culture systems. The first was mouse embryo fibroblast 3T6 cells, in which RL1-negative mutants are impaired and the in vivo phenotype is mimicked. This impairment is amplified when the cells are in the stationary state. The second was mouse embryo testicular carcinoma F9 cells which, in the undifferentiated state, provide a reversal of phenotype; wild-type virus fails to grow but RL1-negative virus replicates efficiently. Differentiation results in the ability to support wild-type virus growth. The stage at which the replication cycle is blocked plus the role of cellular factors is addressed in both tissue culture systems. Evidence is provided that cell type and cell state are crucial to ICP34.5-cellular interaction and hence, based on these parameters, ICP34.5 can be defined as a host-range determinant. Identification of cellular proteins that specifically interact with or are homologues of ICP34.5 may lead to the identification of neuron-specific proteins that have a similar role.