J. Barklie Clements

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.

Temporal regulation of herpes simplex virus type 1 transcription: location of transcripts on the viral genome

Abstract Nuclear and cytoplasmic viral RNAs, synthesized in cells productively infected with herpes simplex virus type 1 at early and late times post-infection and in the presence of DNA and protein synthesis inhibitors, have been analyzed by blot hybridization to viral DNA fragments generated by single and double digests with the Hind III, Bgl II and Hpa I restriction endonucleases. RNA transcribed in the presence of cycloheximide (immediate early, made using a cell RNA polymerase) hybridizes to restricted portions of the HSV-1 genome, and the hybridization patterns of both nuclear and cytoplasmic immediate early RNAs are similar. This finding suggests that synthesis of immediate early RNA within the nucleus may be restricted; alternatively, there may be rapid processing of primary transcripts. Virus-induced protein synthesis is a prerequisite for the switch from the restricted immediate early to the early pattern of transcription in which there is hybridization to all the DNA fragments. The hybridization pattern of RNA labeled in the presence of cytosine arabinoside resembles that of early RNA. Late RNA also hybridizes to all the DNA fragments, but the relative hybridization to the individual DNA fragments is different between early and late times. When late nuclear and late cytoplasmic RNAs are compared, there is greater relative hybridization to several different regions of the genome with nuclear RNA. This implies that either translocation of transcripts to the cytoplasm is regulated or that certain RNAs are degraded more rapidly within the cytoplasm.

The human herpesvirus-8 ORF 57 gene and its properties

Human herpesvirus-8 (HHV-8) is a gamma(2) lymphotropic herpesvirus associated with Kaposis sarcoma, a major neoplasm of AIDS patients, and with other AIDS-related neoplasms. The HHV-8 ORF 57 gene is conserved throughout the herpesvirus family and has a herpes simplex virus type 1 homologue, IE63 (also termed ICP27), which is an essential regulatory protein and acts at both transcriptional and post-transcriptional levels. We show that, contrary to the published HHV-8 sequence, which predicts a protein of 275 amino acids, the ORF 57 gene is spliced, contains a single intron and encodes a protein of 455 amino acids. For several gammaherpesviruses examined, the upstream coding exon is 16-17 amino acids in length and is rich in methionine residues. When ORF 57 was fused to the gene for enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the cellular splicing factor SC-35. Unlike the IE63-EGFP fusion protein, ORF 57-EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, ORF 57-EGFP was capable of shuttling from a transfected monkey nucleus to a recipient mouse nucleus in an interspecies heterokaryon assay. These data indicate that HHV-8 ORF 57 and IE63 possess certain common properties.

Herpes simplex virus induces a processing factor that stimulates poly(A) site usage

Extracts from herpes simplex virus-infected cells and from mock-infected cells have been compared for their ability to process at RNA poly(A) sites in vitro. Nuclear extracts from infected cells contain an activity that increases processing efficiency specifically at a late herpes simplex virus poly(A) site. By contrast, a second virus poly(A) site is processed with equal efficiency by nuclear extracts from infected and mock-infected cells. Using precursor RNAs containing these two virus poly(A) sites in tandem, which allows ready detection of the processing factor, we show that this specific activity is heat labile. Analysis of RNAs produced by virus recombinants that contain the poly(A) site sequences in tandem also indicates that increased processing at the late virus poly(A) site occurs in vivo.

Characterization of transcription-deficient temperature-sensitive mutants of herpes simplex virus type 1.

Abstract Six DNA-negative, temperature-sensitive ( ts ) mutants ( ts B, ts D, ts E, ts K, ts S, and ts T) of herpes simplex virus type 1, which fall into four complementation groups, have been characterized by analyses of the nuclear and cytoplasmic transcripts synthesized in cells infected at the restrictive temperature. Transcripts were hybridized to blot strips containing the separated fragments of virus DNA generated by digestion with restriction endonucleases Hin d III, Hpa I, or Bam H1. The hybridization patterns thus obtained have been compared with the patterns given by RNA isolated from cells infected with wild-type virus and labeled at early and late times postinfection (before and after viral DNA replication), or in the continuous presence of inhibitors of DNA or protein synthesis (Clements, J. B., Watson, R. J., and Wilkie, N. M. (1977). Cell 12 , 275–285). Of these mutants, ts K is the most restricted in transcription and gives a pattern of hybridization similar to that observed with immediate-early RNA (transcribed in the absence of protein synthesis). Two mutants ( ts D and ts T), which lie in a different complementation group to ts K, also give restricted hybridization patterns but express additional regions of the genome as compared with ts K RNA. Inasmuch as these three mutants fall into two complementation groups, these experiments suggest that at least two viral products are required to progress from the immediate-early to the early stage of transcription. The hybridization patterns obtained with the other early mutants examined here ( ts B, ts E, and ts S) are, in contrast, much less restricted, and resemble the early, rather than late pattern. Downshift of ts K-infected cells from the nonpermissive to the permissive temperature, both in the presence and absence of further protein synthesis, results in the transcription of regions which map throughout the viral genome. These experiments suggest, therefore, that the polypeptides which are required for the switch-on of these additional transcripts accumulate in ts K-infected cells under restrictive conditions.

Direct Stimulation of Translation by the Multifunctional Herpesvirus ICP27 Protein

ABSTRACT Herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential regulator of viral gene expression with roles at various levels of RNA metabolism in the nucleus. Using the tethered function assay, we showed a cytoplasmic activity for ICP27 in directly enhancing mRNA translation in vivo in the absence of other viral factors. The region of ICP27 required for translational stimulation maps to the C terminus. Furthermore, in infected cells, ICP27 is associated with polyribosomes, indicating a function in translation during the lytic cycle.

CK2 Protein Kinase Is Stimulated and Redistributed by Functional Herpes Simplex Virus ICP27 Protein

ABSTRACT It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic α or α′ subunits and two regulatory β subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.

The Human Papillomavirus Type 31 Late 3′ Untranslated Region Contains a Complex Bipartite Negative Regulatory Element

ABSTRACT The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3′ untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF65, and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element.

Functional Domains within the Nucleus of a Cell Infected with HSV-1

HSV‐1 is a nuclear replicating DNA virus capable of establishing both lytic and latent infections in mammalian cells. Expression of the more than 80 HSV genes (the majority of which do not contain introns) requires complex coordination of viral and cellular factors both temporally, at appropriate points during the infectious cycle, and spatially as the virus transcription, replication and DNA packaging factories develop in the cell nucleus. Whilst the HSV genome encodes sufficient proteins to sustain viral DNA replication, it is reliant upon its host cell for RNA polymerase II and RNA processing machinery, in addition to an increasing number of cellular cofactors, for gene expression.

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).