Herpes simplex virus type 1 (HSV-1), the prototype ␣-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 ␤ 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 antihnRNP 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.The involvement of herpesviruses in a range of prominent medical or veterinary diseases makes them one of the most important virus families. Herpes simplex virus type 1 (HSV-1), 1 a common and effective human pathogen, is capable of establishing both lytic and latent infectious life cycles, and up to 80% of adults in the developed world are seropositive for this virus. HSV-1 is a nuclear replicating virus with a large doublestranded DNA genome that encodes some 80 gene products (1). During lytic infection, virus genes are expressed in a temporal cascade and are categorized as immediate early (IE, ␣), early (␤), or late (␥) based on the time postinfection of their expression (2). The five IE gene products, which do not require prior viral protein synthesis for their expression, regulate early and late gene expression and subvert the host cytotoxic T-lymphocyte response (3, 4). A key IE protein is the 63-kDa IE phosphoprotein IE63 also called ICP27 (5). IE63 is one of two HSV IE proteins essential for lytic virus replication (6) and is the only regulatory gene with a homologue in every herpesvirus of mammals and birds sequenced so far (7), suggesting that aspects of its regulatory role are maintained throughout the herpesvirus family.Studies of IE63 have shown that its expression is required for the switch from early to late virus gene expression (8) and have highlighted the multifunctional nature of this protein that acts both at the transcriptional and post-transcriptional levels (reviewed in Ref. 9). Acting post-transcriptionally, IE63 binds RNA in vivo with a reported specificity for intronless viral transcripts (10), enhances pre-mRNA 3Ј processing (11), in...

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.

SUMMARYThe 5' ends of two early herpes simplex virus type I mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0-60, are unspliced and share a 3' terminus. Genomic DNA at the 5' ends has been sequenced and the 5' termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5' end of the 1-2 kb mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap.

We have constructed a series of random N-terminal deletions of the large subunit (R1) of the herpes simplex virus type 1 ribonucleotide reductase. Deletions extended throughout the R1 gene open reading frame and, in total, 31 different truncated polypeptides were expressed in Escherichia coli using the T7 expression system. N-Terminal truncations were analyzed for their interaction with the small subunit (R2) of ribonucleotide reductase using a sensitive enzyme-linked immunosorbent assay (ELISA) method and for their ability to complement R2 in ribonucleotide reductase assays. Truncated proteins were also tested for homodimerization using gel-filtration chromatography. The results identified a region of R1 between amino acids 349 and 373 which was essential for subunit interaction. Proteins lacking up to 348 amino-terminal residues associated with R2 and complemented R2 in ribonucleotide reductase assays. Proteins commencing at amino acid 373 and beyond did not interact with R2 and were inactive in enzyme assays. Using a plasmid which expressed an N-terminal deleted protein commencing at amino acid 247, we constructed two defined C-terminal deletions to give proteins comprising amino acids 247-434 and 247-996 of R1. Neither of these truncated proteins bound R2 and we concluded that a second region between amino acids 996 and 1137 (the C-terminus) is required for interaction with R2. Gel-filtration studies indicated that deletion of the first 420 amino acids from R1 did not affect dimerization. However, deletions of 457 amino acids and larger gave proteins which existed as monomers.(ABSTRACT TRUNCATED AT 250 WORDS)