Forced degradation of Fas inhibits apoptosis in adenovirus-infected cells.

DNA viruses have evolved elaborate mechanisms to overcome host antiviral defences. In adenovirus-infected cells, programmed cell death (apoptosis) induced by the cytokine tumour necrosis factor (TNF) is inhibited by several adenovirus-encoded proteins. Occupation of the cell-surface receptor Fas, a member of the TNF-receptor superfamily that is expressed on most cell types, triggers apoptosis of that cell. Here we show that the adenovirus RID (for receptor internalization and degradation) protein complex, which is an inhibitor of TNF-induced apoptosis, mediates internalization of cell-surface Fas and its destruction inside lysosomes within the cell. Fas has not previously been shown to be internalized and then degraded. RID also mediates internalization of the receptor for epidermal growth factor, but it does not affect the transferrin receptor or class I antigens of the major histocompatibility complex. Removal of Fas from the surface of adenovirus-infected cells expressing RID may allow infected cells to resist Fas-mediated cell death and thus promote their survival.

Equine herpesvirus-1 strain KyA, a candidate vaccine strain, reduces viral titers in mice challenged with a pathogenic strain, RacL.

The equine herpesvirus type-1 (EHV-1) strain Kentucky A (KyA) has a long history of repeated passage either in vivo in the Syrian hamster or in vitro in mouse L-M fibroblast tissue culture. This repeated passage in cells other than those of the natural host has caused genomic alterations of the KyA chromosome resulting in deletion of several genes or portions of open reading frames (ORFs). This report presents in vivo data from a mouse model of EHV-1 infection demonstrating the attenuated nature of EHV-1 strain KyA and that intranasal infection with KyA protects animals from subsequent challenge with a pathogenic strain, RacL, by reducing RacL viral titers in the lungs of the challenged animals. Mice infected with KyA exhibit no clinical manifestations of EHV-1 disease and do not experience the wasting that occurs with RacL infection. KyA-infected mice clear virus from the lung by day 5 post-infection (p.i.), whereas RacL infected mice have substantial virus titers (5 x 10(5) pfu/lung) at this time point. Intranasal infection with KyA followed by a challenge with RacL 4 weeks post-KyA infection resulted in a significant (P = 0.0079) reduction in the lung titers of the RacL virus. RacL was identified as the virus present in the lungs of the challenged mice by a PCR assay employing primers to amplify the EUS4 gene which differs in size by 1.2 kilobase pairs (kbp) in the two strains. Importantly, the protection afforded by KyA is long lasting in that challenge with RacL 15 months after KyA infection, results in reduced virus titers and viral clearance by day 5 post-challenge. These results support the further consideration of EHV-1 KyA as a live virus vaccine.

Localization of the Us protein kinase of equine herpesvirus type 1 is affected by the cytoplasmic structures formed by the noval IR6 protein.

Previous work revealed that the Us (unique short) segment of equine herpesvirus type-1 (EHV-1), like that of other alphaherpesviruses, encodes a serine/threonine protein kinase (PK). Experiments were carried out to identify the PK encoded by the EHV-1 EUS2 gene (ORF 69) and to ascertain its time course of synthesis and cellular localization. Western blot and immunoprecipitation analyses of EHV-1-infected cell extracts using a PK-specific polyclonal antibody generated against a bacterially expressed TrpE/PK fusion protein identified the Us PK as a 42- to 45-kDa phosphoprotein. The PK protein is first synthesized at 3 hr postinfection, is produced throughout the infection cycle, and is incorporated into EHV-1 virions. Interestingly, immunoprecipitation analyses revealed that the PK protein within the cytoplasm is associated with the 33-kDa IR6 novel protein of EHV-1, is expressed abundantly as an early protein, and is present in the large rod-like structures formed by the IR6 protein (ORF67 protein) within the cytoplasm of infected cells. Confocal microscopic examination of cells stained with fluorescein-labeled antibody clearly showed that the PK protein colocalized with the cytoplasmic IR6 rod-like structures and remained associated with these unique structures during infection. In contrast, in cells infected with the EHV-1 RacM strain in which the IR6 protein harbors four amino acid substitutions that prevent formation of the rod-like structures (Osterrieder et al., 1996, Virology 217, 442-451), the PK protein localized predominantly to the nucleus. The possible significance of the association of the IR6 and PK proteins in EHV-1 replication is discussed.

Transcriptional analyses of the unique short segment of EHV-1 strain Kentucky A.

The unique short (Us) segment of the genome of equine herpesvirus type 1 (EHV-1) strain KyA is comprised of six open reading frames (ORFs) that encode: a) a homolog of the Us2 protein of herpes simplex virus type 1 (HSV-1); b) a serine threonine protein kinase that is a homolog of the HSV-1 Us3 protein; c) a homolog of pseudorabies virus glycoprotein gX and HSV-2 gG; d) a novel glycoprotein, EUS4, not encoded by other herpesviruses sequenced to date; e) a homolog of HSV-1 gD; and f) a homolog of HSV-1 Us9. The KyA strain is a deletion mutant that lacks Us sequences encoding gI, gE, and a potential 10 kD polypeptide, and thus may be useful as a parent virus for the generation of live virus vaccines. To complete the elucidation of the transcriptional program of the Us segment, Northern blot hybridization and S1 nuclease analyses were performed on poly(A)(+)-selected RNA isolated from infected cells maintained under early (phosphonoacetic acid-block) and late conditions. The findings revealed that the gene (EUS2 ORF) encoding the protein kinase is expressed as an early 2.9 kb transcript that overlaps and is 3' coterminal with a 1.6 kb early transcript that encodes the gG/gX homolog (EUS3 ORF). Two transcripts of 1.6 kb and 5.8 kb are 5' coterminal and may both encode the novel glycoprotein gene EUS4. The 1.6 kb transcript terminates at a poly(A) signal site downstream of the EUS4 ORF, and the 5.8 kb transcript terminates within the inverted repeat (IR) segment. Overall, the transcriptional program of the EHV-1 KyA Us segment is complex and exhibits similarities to that of HSV-1 Us segment: a) transcripts arise from both DNA strands; b) some transcripts, including those mapping at the termini of the Us segment, extend into the IR segments and are 3' coterminal with the 1.2 kb IR6 transcript; c) at least one transcript reads through a functional polyadenylation signal; d) some transcripts encoding genes that lie in different reading frames exist as a family of overlapping mRNAs, some in an anti-sense manner. Lastly, of the six Us genes of the EHV-1 KyA strain, only those encoding the EHV-1 protein kinase and the HSV-2 gG/gX homolog are members of the early kinetic class.

Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.

Identification and transcriptional mapping of genes encoded at the IR/Us junction of equine herpesvirus type 1.

Two open reading frames (ORFs) encoded at the inverted repeat unique short (Us) junction of the Short (S) region of the equine herpesvirus type 1 genome were identified by DNA sequencing of a 2876 base pair (bp) genomic segment, and transcripts encoding these ORFs were characterized by Northern blot, S1 nuclease, and primer extension analyses. These studies also established the size of each inverted repeat to be 12,768 nucleotides (nts). The IR6 ORF (816 bp), mapping at nts 12,317-11,502 of the S region, is the last gene completely encoded within each inverted repeat and encodes a predicted 30.1-kDa protein of 272 amino acids, which does not exhibit homology to other alphaherpesvirus proteins. IR6 is expressed as an early transcript of 1.2 kb which is detected initially at 1.5 hr p.i. and up to 12 hr p.i. The transcription initiation and termination sites of IR6 were mapped by primer extension and S1 nuclease analyses to nts 12,465 and 11,408, respectively. The first ORF encoded within the Us segment (909 bp; EUS1), mapping at nts 13,397-12,489, encodes a predicted 33.5-kDa protein of 303 amino acids that exhibits 29% identity to the US2 protein of herpes simplex virus 1. EUS1 is expressed as a 2.3-kb mRNA of the gamma-1 class, as its synthesis begins prior to viral DNA replication at 4 hr p.i. but is retarded by phosphonoacetic acid, an inhibitor of viral DNA replication. The Tci and Tct sites of EUS1 were mapped by S1 nuclease analyses to nts 13,637 and 11,408, respectively. Interestingly, this termination site is also utilized by three late mRNAs of 5.8, 3.8, and 1.7 kb which originate within the Us and overlap the IR6 mRNA encoded in the terminal inverted repeat (TR) of the prototype genomic isomer. EUS1 is 3' coterminal with IR6 in the inverted repeat, whereas, the 5.8, 3.8, and 1.7 kb transcripts are 3' coterminal with IR6 of the TR.

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.

Mapping the termini and intron of the spliced immediate-early transcript of equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) has been shown to synthesize a 6.0-kilobase (kb) species of immediate-early (IE) mRNA in productively infected cells. This IE gene region maps within the outer portion (map units 0.79 to 0.83 and 0.96 to 1.00) of the two inverted repeat segments of the short genomic region, and elucidation of its DNA sequence has revealed multiple potential open reading frames (ORFs), including a major ORF of 4,461 nucleotides (F. J. Grundy, R. P. Baumann, and D. J. O'Callaghan, Virology 172:223-236, 1989). Analyses of IE polypeptides synthesized in EHV-1-infected cells (in vivo) and in vitro translation of hybrid-selected IE mRNA indicated that multiple species of IE proteins are encoded by this IE mRNA species. To address the nature of the 6.0-kb IE RNA species, Northern (RNA) blot hybridization, S1 nuclease mapping, and primer extension analyses have been employed. These data revealed that no major introns were detected within the body of the IE transcript. However, the IE mRNA was shown to be spliced at the 5' terminus, such that a 372-base intron containing two small ORFs (19 and 51 amino acids) was removed from the leader region of the transcript. This splicing event reduced the leader region from 625 to 253 bases. S1 and primer extension analyses of the 5' terminus of this transcript revealed that the transcription initiation site is located 24 to 26 bases downstream of the consensus TATAAA motif. The 3' transcription termination site was mapped by S1 nuclease analysis to approximately 10 to 20 bases downstream of the polyadenylation signal, AATAAA. The distance from the stop codon of the major ORF to the polyadenylation site is approximately 300 bases. Results from S1 nuclease experiments indicated that splicing does not occur at the 3' terminus. These studies indicated that the EHV-1 6.0-kb IE mRNA is spliced at the 5' terminus and that alternative splicing of this transcript may function in regulating translation of the IE mRNA species.