Transcriptional and translational analyses of the UL2 gene of equine herpesvirus 1: a homolog of UL55 of herpes simplex virus type 1 that is maintained in the genome of defective interfering particles

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The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 ␣ regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides ؊123 to ؉20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.

Section: Methodsmentioning

confidence: 99%

Regulatory function of the equine herpesvirus 1 ICP27 gene product

Zhao

Holden

Smith

et al. 1995

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“…Recently, the UL2 gene of EHV-1 (a homolog of UL55 of herpes simplex virus type 1 [HSV-1]), which is conserved in the DIP genome, was shown to direct the synthesis of a 0.9-kb mRNA and a 23-kDa polypeptide that are overexpressed in DIP-enriched infection (18). In addition to UL2 and sequences that contain the origin of replication and the cleavage-packaging elements (50), other sequences conserved in the DIP genome were shown to generate a hybrid gene composed of sequences that encode the N-terminal portion of the IR4 protein (ICP22 homolog [23]) joined to sequences encoding the C-terminal portion of the UL3 protein (ICP27 homolog [53]).…”

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confidence: 99%

Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1

Harty

Caughman

Holden

et al. 1993

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Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The ULl gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. ULI has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 ULl gene belongs to the early kinetic class and is transcribed as a 1.2-kb polyadenylated mRNA (R.

“…Although the function of UL2 has not been elucidated, the homologous UL55 gene of HSV-1 may be involved in inhibition of both virus gene expression and transformation (7). Both the UL1 and UL2 transcripts and proteins are produced in large amounts in DI particle-enriched infections (19,20), and an additional 2.2-kb transcript has been detected only EHV-1 DI particle-enriched infection (data not shown). The 2.2-kb transcript has not been characterized in detail, but preliminary data revealed that the bulk of this transcript lacks a poly(A) tail and overlaps approximately 700 nt of the 3Ј end of the immediateearly mRNA (21a).…”

Section: Discussionmentioning

confidence: 94%

“…To date, no function has been proposed for UL1 or its homologs. The UL2 gene, a homolog of UL55 of HSV-1 and ORF3 of varicella-zoster virus, was shown to express a 23-kDa protein in both EHV-1 STD infection and DI particle-enriched infection (20,65). Although the function of UL2 has not been elucidated, the homologous UL55 gene of HSV-1 may be involved in inhibition of both virus gene expression and transformation (7).…”

Section: Discussionmentioning

confidence: 99%

“…Infected cell lysates or immunoprecipitated protein samples were boiled with Laemmli sample buffer for 5 min and fractionated through 5% stacking and 10% resolving SDS-polyacrylamide gel electrophoresis (PAGE) as described previously (19)(20)(21). Proteins in the gel were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, N.H.) at 20 V overnight.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection

Chen

Harty

Zhao

et al. 1996

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J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.