High baseline bilirubin and low albumin predict liver decompensation and serious adverse events in HCV-infected patients treated with sofosbuvir-containing regimens.

To conduct surveillance and determine the safety profile of new hepatitis C virus treatments in real-world clinical practice. Hepatic decompensation and other serious adverse events were investigated in an observational cohort study of 511 patients treated with regimens containing sofosbuvir, December 2013-June 2014. Among 499 previously stable patients (no history of hepatic decompensation during the previous 12 months), a nested case-control study was performed to identify predictors of decompensation/serious adverse event. Cases and controls were matched 1:5 based on treatment regimen and duration. Matched conditional logistic regression was used for analysis. Providers scored the likelihood that events were treatment-related (scale = 0-4). The cumulative incidence of decompensation/events was 6.4% for the total cohort. Among 499 previously stable patients, the incidence of decompensation/events was 4.5%; the mortality rate was 0.6%. Sixteen of the 499 experienced one or more serious complications considered to be at least potentially treatment-related, and the sustained virological response rate was 7/16 (44%). Two cases, both on sofosbuvir/simeprevir (without interferon or ribavirin), had complications consistent with autoimmune events (score 3, 'likely treatment-related'), and one experienced a flare of autoimmune hepatitis. Compared to controls, cases had higher baseline median model for end-stage liver disease scores (14 vs 8, P < 0.01). Decompensation/events was independently associated with lower baseline albumin (OR = 0.12/g/dL, P = 0.01) and higher total bilirubin (OR = 4.31/mg/dL, P = 0.01). Reduced hepatic function at baseline increased the risk of liver decompensation/events.

The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE.

Epstein-Barr virus nuclear antigen 2 (EBNA 2) activates transcription of specific genes and is essential for B-lymphocyte transformation. EBNA 2 has an acidic activation domain which interacts with general transcription factors TFIIB, TFIIH, and TAF40. We now show that EBNA 2 is specifically bound to a novel nuclear protein, p100, and that p100 can coactivate gene expression mediated by the EBNA 2 acidic domain. The EBNA 2 acidic domain was used to affinity purify p100. cDNA clones encoding the p100 open reading frame were identified on the basis of peptide sequences of the purified protein. Antibody against p100 coimmunoprecipitated p100 and EBNA 2 from Epstein-Barr virus-transformed lymphocyte extracts, indicating that EBNA 2 and p100 are complexed in vivo. p100 overexpression in cells specifically augmented EBNA 2 acidic domain-mediated activation. The coactivating effect is probably mediated by p100 interaction with TFIIE. Bacterially expressed p100 specifically adsorbs TFIIE from nuclear extracts, and in vitro-translated p56 or p34 TFIIE subunit can independently bind to p100. p100 also appears to be essential for normal cell growth, since cell viability was reduced by antisense p100 RNA and restored by sense p100 RNA expression.

Sequence and organization of the genomic termini of equine herpesvirus type 1.

The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DR1-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the 'a' sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV 'a' sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DR1-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DR1-Uc (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/packaging elements of other herpesviruses are discussed.

Equine herpesvirus 1 sequence near the left terminus codes for two open reading frames.

We have previously reported the sequence of the equine herpesvirus one genomic termini that are homologous to the genomic termini of other herpesviruses. In this paper, we present the nucleotide sequence adjacent to the left terminus sequence (map units 0.0087 to 0.0237). This sequence codes for two open reading frames (ORF) which are homologous to ORF2 and ORF3 of the varicella-zoster virus genome and are located at colinear positions. The L region sequence presented here also contains a segment that is involved in the generation of the genome of EHV-1 DI particles through recombination with sequences mapping within the internal portion of the inverted repeat sequences of the short region.

The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene.

The complete nucleotide sequence of the inverted repeat component (IR; 12,776 bp each) of the genome of equine herpesvirus type 1 (EHV-1) has been determined. Transcription analyses have revealed that the EHV-1 IR sequence encodes at least 6 genes. In this report, we present the DNA sequence and transcriptional characterization of a gene (IR3) that maps entirely within the IR sequences. The IR3 open reading frame (ORF) is located between nucleotides (nt) 6123-6411 of the IR sequence and possesses an ORF of 95 amino acids. Interestingly, this ORF does not show homology to any known herpesvirus gene, suggesting that the IR3 gene is unique to EHV-1. Moreover, the location of the IR3 gene between the immediate-early (IR1) gene and the origin of replication is unique in comparison to the IR gene arrangement of other alphaherpesviruses such as herpes simplex virus type 1 and varicella zoster virus. Putative cis-acting elements flanking the IR3 ORF include a TATA box (nt 5648-5652), a GC box (nt 5600-5605), and three polyadenylation signals (nt 6533-6538, 6648-6653, and 6663-6668). Northern blot analyses identified a 1.0 kb mRNA that exhibits characteristics of a late gene of the gamma-1 class. Northern blot, S1 nuclease, and primer extension analyses revealed that transcription of IR3 initiates within the intron of the immediate-early gene (IR1) on the opposite stand of the genome. Thus, the 5' end of IR3 transcript is antisense to the 5' end of the IR1 mRNA and promoter, and IR3 transcription may regulate the expression of IR1 during late times of infection.

Recurrent ergotism: a case report.

A patient was seen with absent pulses and vague symptoms. History and clinical findings supported a diagnosis of ergotism due to heavy use of ergotamine suppositories. The patient continued to use ergotamine despite warnings and was seen on two additional occasions with ergotism. Previously a heavy user of ergotamine, she became sensitive to small doses, demonstrating marked vasospasm after only one suppository. This phenomenon, not previously reported, is postulated secondarily to drug interaction. The patient is presented with a review of the pharmacology, toxicity, and treatment of ergotamine-induced ergotism. Alerted to the possibility of drug interactions, physicians may safely use ergotamine in the treatment of migraine headache with careful monitoring for signs and symptoms of early toxicity.

Toward unsupervised classification of calcified arterial lesions.

There is growing evidence that calcified arterial deposits play a crucial role in the pathogenesis of cardiovascular disease. This paper investigates the challenging problem of unsupervised calcified lesion classification. We propose an algorithm, US-CALC (UnSupervised Calcified Arterial Lesion Classification), that discriminates arterial lesions from non-arterial lesions. The proposed method first mines the characteristics of calcified lesions using a novel optimization criterion and then identifies a subset of lesion features which is optimal for classification. Second, a two stage clustering is deployed to discriminate between arterial and non-arterial lesions. A histogram intersection distance measure is incorporated to determine cluster proximity. The clustering hierarchies are carefully validated and the final clusters are determined by a new intracluster compactness measure. Experimental results indicate an average accuracy of approximately 80% on a database of electron beam CT heart scans.

Residues 231 to 280 of the Epstein-Barr virus nuclear protein 2 are not essential for primary B-lymphocyte growth transformation.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is a transcriptional transactivator of cellular and viral gene expression and is essential for the transformation of resting human B lymphocytes into long-term lymphoblastoid cell lines (LCLs). Previous molecular genetic analyses identified three domains that are critical for transformation and showed that the rest of EBNA-2 is not critical. We now find that codons 231 to 280 that were part of one of the critical domains (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991) can be deleted with only a small effect on the ability of EBNA-2 to transactivate gene expression. In transient transfection assays, EBNA-2 deleted for codons 231 to 280 accumulated to higher levels and was similar to wild-type EBNA-2 in activation of the BamC promoter and in association with RBPJk, a cellular transcription factor that is important for EBNA-2 interaction with promoter regulatory elements. However, EBNA-2 d231-280 activated the viral latent membrane protein 1 (LMP1) promoter with only 60% of wild-type efficiency. Recombinant EBVs specifically deleted for EBNA-2 codons 231 to 280 were efficient in initiating the transformation of resting primary human B lymphocytes into LCLs. However, these LCLs grew less well than wild-type EBV-transformed LCLs, and 4- to 10-fold more cells were required for outgrowth following limit dilution. EBNA-2 d231-280 accumulated to unusually high levels in the recombinant transformed LCLs, and this was associated with somewhat higher EBNA-1 and lower LMP1 expression, consistent with the near-wild-type activation of the BamC EBNA promoter and the abnormally low activation of the LMP1 promoter in transient transfection assays. Thus, EBNA-2 d231-280 modestly perturbed the regulation of viral gene expression and resulted in less LMP1, while having surprisingly subtle effects on LCL outgrowth. Deletion of EBNA-2 codons 292 to 310, which are closer to the site that specifies interaction with RBPJk, was more disruptive of RBPJk association and of the ability to transform B lymphocytes.

Epstein-Barr virus nuclear protein 2 has at least two N-terminal domains that mediate self-association.

Previous genetic and biochemical analyses have indicated that the Epstein-Barr virus EBNA-2 amino terminus is important for primary B-lymphocyte growth transformation and may be involved in self-association. We now report that EBNA-2 has at least two domains, amino acids 1 to 60 and 96 to 210, which independently mediate homotypic association, 1 to 60 with 1 to 60 and 96 to 210 with 96 to 210. EBNA-2 self-association is likely to be critical to the ability of EBNA-2 to interact simultaneously with multiple cellular transcription factors, coactivators, and histone acetyltransferases through its RBPJkappa binding and acidic activating domains.

The N-terminal half of EBNA2, except for seven prolines, is not essential for primary B-lymphocyte growth transformation.

Previous molecular genetic analyses of Epstein-Barr virus nuclear protein 2 (EBNA2) identified a negative effect of deletion of codons 19 to 33 on transformation and gene transactivation, while deletion of codons 19 to 110 was a null mutation for transformation and gene transactivation. We here report the surprising finding that codons 2 to 88, which encode the highly conserved unique N terminus (amino acids 1 to 58) and most of the polyproline repeat (amino acids 59 to 95), can be deleted with only minimal effects on transformation. Codons 97 to 122 can also be deleted with only minimal effects on transformation. However, deletion of 35 of the 37 prolines (amino acids 59 to 93) or deletion of codons 2 to 95 results in a null transforming phenotype. Although EBNA2 from which codons 59 to 93 were deleted was a null mutation for transformation, it was similar to some transforming mutants of EBNA2 in abundance, in interaction with RBPJK, and in transactivation of the LMP1 promoter in transient transfection assays. These data indicate that between three and seven prolines are critical for EBNA2 structure or for intermolecular interaction. Aside from these seven prolines, codons encoding the rest of the N-terminal half (amino acids 2 to 230) of EBNA2 are nonessential for primary B-lymphocyte growth transformation.

The EBNA-2 arginine-glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.

Organization and function of the ORIs sequence in the genome of EHV-1 DI particles.

Equine herpesvirus type 1 (EHV-1) cultures enriched for defective interfering particles (DIP) mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. We have recently demonstrated that an origin of replication (ORI) is located within the central portion (map units 0.828 and 0.948) of the inverted repeat sequence (IRs) of the short region of the standard EHV-1 genome. In the generation of the genome of EHV-1 DI particles, sequences from this internal portion of the IRs recombine with sequences at the long region terminus at nucleotides 3244-3251. In this paper we report that the ORIs sequence is precisely conserved in the DIP genome, that direct repeat sequences near the ORIs sequence which may enhance DNA replication are mutated in the DIP genome, and that the ORI sequence of DIP DNA is functional in DNA replication assays.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors.

Since Epstein-Barr virus (EBV) infection of Burkitt's lymphoma (BL) cells in vitro reproduces many of the activation effects of EBV infection of primary B lymphocytes, mRNAs induced in BL cells have been cloned and identified by subtractive hybridization. Nine genes encode RNAs which are 4- to > 100-fold more abundant after EBV infection. Two of these, the genes for CD21 and vimentin, were previously known to be induced by EBV infection. Five others, the genes for cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, and the myristylated alanine-rich protein kinase C substrate (MARCKS), are genes which were not previously known to be induced by EBV infection. Two novel genes, EBV-induced genes 1 and 2 (EBI 1 and EBI 2, respectively) can be predicted from their cDNA sequences to encode G protein-coupled peptide receptors. EBI 1 is expressed exclusively in B- and T-lymphocyte cell lines and in lymphoid tissues and is highly homologous to the interleukin 8 receptors. EBI 2 is most closely related to the thrombin receptor. EBI 2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. EBI 2 is also expressed at lower levels in a promyelocytic and a histiocytic cell line and in pulmonary tissue. These predicted G protein-coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection.

Transcriptional analysis of the UL1 gene of equine herpesvirus 1: a gene conserved in the genome of defective interfering particles.

Defective interfering particles (DIPs) of equine herpesvirus type 1 (EHV-1) are biologically active, in that they mediate the coestablishment of oncogenic transformation and persistent infection in permissive, primary hamster embryo fibroblasts. The DIP genome is composed of EHV-1 sequences originating from the L-terminus (mapping units (m.u.) 0.00-0.023), the junction of the unique long (UL) region and the internal inverted repeat (IR) (m.u. 0.78-0.79 and 0.99-1.00), and the central portion of the IR (m. u. 0.83-0.87 and 0.91-0.95). The nature of one of the genes (UL1) mapping at the L-terminus was analyzed at the RNA level by Northern blot hybridization and S1 nuclease analyses. These data, and DNA sequencing analyses reported previously revealed that the UL1 gene: (1) contains a major open reading frame (ORF) of 258 amino acids, (2) is a homologue of the ORF2 gene of varicella zoster virus (VZV), (3) is conserved in the genome of DIPs of EHV-1, (4) encodes a 1.2-kb early (E) mRNA that is transcribed toward the short region of the genome, (5) utilizes a transcription initiation site approximately 1,120 nucleotides from the L-terminus, and (6) utilizes a transcription termination site approximately 2211 nucleotides from the L-terminus. These initial studies serve as the basis of future work to determine the function of the UL1 gene in cytolytic infection, and its potential role in EHV-1 persistent infection.

Genetic and biochemical evidence that EBNA 2 interaction with a 63-kDa cellular GTG-binding protein is essential for B lymphocyte growth transformation by EBV.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA 2) is an acidic transcriptional transactivator of virus and cell gene expression and is essential for growth transformation of primary B lymphocytes. EBNA 2 transactivation of response elements (E2REs) can be mediated by interaction with a GTGGGAA-specific DNA-binding factor(s). We now purify the factor by S-Sepharose and EBNA 2 affinity chromatography and identify it as a single 63-kDa protein. The protein is shown to specifically coimmunoprecipitate with EBNA 2 from lymphoblasts transfected with an EBNA 2 FLAG expression vector. Mutation of GTG to TCT in a GTGGGAA motif common to the Cp, LMP2, and LMP1 promoters results in loss of recognition by p63. EBNA 2 amino acids 310-336 are sufficient for p63 binding. The only motif in this 27 amino acid sequence which is common to the EBNA 2 genes of EBV types 1 and 2 is GPPWWPP (I/V) (C/R) DP, which is therefore likely to mediate p63 interaction. Mutation of WW to SS or FF ablates interaction with p63, indicating that both the hydrophobic and aromatic characteristics of WW are essential for its "key" interaction with p63. EBNA 2 with a WW mutated to SS is also unable to marker rescue primary B lymphocyte transforming virus from cells infected with an EBNA 2-deleted virus, while otherwise isogenic wild-type EBNA 2 readily marker rescues transforming virus in parallel experiments. EBNA 2 transactivation through the Cp E2RE is completely abolished by the WW to SS mutation while transactivation of -234 to +40 LMP1 E2RE is only partially affected. These genetic and biochemical experiments support the hypothesis that EBNA 2 WW interaction with a p63 GTGGGAA-binding protein is essential for EBV-mediated cell growth transformation because it specifically associates EBNA 2 with its response elements. This enables the EBNA 2 acidic domain to transcriptionally transactivate specific genes.

The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family.

The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family.

ICP22 homolog of equine herpesvirus 1: expression from early and late promoters.

The complete nucleotide sequence of the short region, made up of a unique segment (Us; 6.5 kb) bracketed by a pair of inverted repeat sequences (IR; 12.8 kb each), of the equine herpesvirus 1 (EHV-1) genome has been determined recently in our laboratory. Analysis of the IR segment revealed a major open reading frame (ORF) designated IR4. The IR4 ORF exhibits significant homology to the immediate-early gene US1 (ICP22) of herpes simplex virus type 1 and to the ICP22 homologs of varicella-zoster virus (ORF63), pseudorabies virus (RSp40), and equine herpesvirus 4 (ORF4). The IR4 ORF is located entirely within each of the inverted repeat sequences (nucleotides [nt] 7918 to 9327) and has the potential to encode a polypeptide of 469 amino acids (49,890 Da). Within the IR4 ORF are two reiterated sequences: a 7-nt sequence tandemly repeated 17 times and a 25-nt sequence tandemly repeated 13 times. Nucleotide sequence analyses of IR4 also revealed several potential cis-regulatory sequences, two TATA sequences separated by 287 nt, an in-frame translation initiation codon following each TATA sequence, and a single polyadenylation site. To address the nature of the mRNA species encoded by IR4, we used Northern (RNA) blot and S1 nuclease analyses. RNA mapping data revealed that IR4 has two promoters that are regulated differentially during a lytic infection. A 1.4-kb mRNA appears initially at 2 h postinfection and is an early transcript since its synthesis is not affected by the presence of phosphonoacetic acid, an inhibitor of EHV-1 DNA replication. In contrast, a 1.7-kb mRNA appears at later times postinfection and is designated as a gamma-1 transcript, since its synthesis is significantly reduced by phosphonoacetic acid. These IR4-specific mRNAs are 3' coterminal, have unique 5' termini, and would code for in-frame, overlapping, carboxy-coterminal proteins of 293 and 469 amino acids, respectively. Interestingly, the site of homologous recombination to generate the genome of EHV-1 defective interfering particles that initiate persistent infection occurs between nt 3244 and 3251 of UL3 (ICP27 homolog) and nt 9027 and 9034 of IR4 (ICP22 homolog). Thus, this recombination event would generate a unique ORF that would encode a potential protein whose amino end was derived from the N-terminal 193 amino acids of the ICP22 homolog and whose carboxyl end was derived from the C-terminal 68 amino acids of the ICP27 homolog.

Identification and characterization of an equine herpesvirus 1 late gene encoding a potential zinc finger.

In this report, we present the DNA sequence and transcriptional characterization of a gene (IR5) that maps within each of the inverted repeat (IR) segments of the equine herpesvirus type 1 (EHV-1) genome. The IR5 open reading frame (ORF) is located within both IR sequences (nucleotides 9932-10,642 of the IR). DNA sequence analyses of the IR5 gene region revealed an ORF of 236 amino acids (24,793 Da) that showed significant homology to ORF64 of varicella-zoster virus and ORF3 of EHV-4 both of which map within the inverted repeats and to the US10 ORF of herpes simplex virus type 1 (HSV-1) which maps within the unique short segment. Additional analyses of the nucleotide sequence failed to reveal any overlapping ORFs that would correspond to US11 or US12 of HSV-1. Interestingly, the IR5 ORF of EHV-1 possesses a sequence of 13 amino acids (CAYWCCLGHAFAC) that is a perfect match to the consensus zinc finger motif (C-X2-4-C-X2-15-C/H-X2-4-C/H). Putative cis-acting elements flanking the IR5 ORF include a TATA box (nucleotides 9864-9870), a CAAT box (nucleotides 9709-9714), and a polyadenylation signal (nucleotides 10,645-10,650). Northern blot and S1 nuclease analyses identified a single 0.9-kb mRNA species that first appears at 2 hr postinfection, and whose synthesis is reduced in the presence of phosphonoacetic acid, an inhibitor of EHV-1 DNA synthesis. Thus, the IR5 gene of EHV-1 exhibits characteristics representative of a late gene of the gamma-1 class. The characterization of the IR5 gene at the DNA and RNA levels will facilitate ongoing studies to identify and characterize the IR5 polypeptide.