The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

Abstract

The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed.

The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters

“…Cells were transfected using lipofectin (Invitrogen) and Opti-MEM medium (Gibco, BRL, Carlsbad, CA) as detailed elsewhere (Ahn et al, 2007). Briefly, 1 pmol of the various reporter plasmids and 0.5 pmol of the expression plasmids were co-transfected into RK13 cells.…”

“…The early IR2 protein is a truncated form of the IE protein (Harty and O’Callaghan, 1991) and serves to down-regulate the expression from all classes of viral promoters (Kim et al, 2006). Lastly, the EHV-1 unique IR3 gene encodes a transcript that is antisense to the IE mRNA (Holden et al, 1992), is not translated to a detectable protein product (Ahn et al, 2007), and functions to down-regulate expression of the IE gene (Ahn et al, 2010). …”

The early UL3 gene of equine herpesvirus-1 encodes a tegument protein not essential for replication or virulence in the mouse

“…Luciferase reporter assays were performed as previously described (Ahn et al, 2007). Briefly, 80% confluent RK13 cells were prepared in 24 well plates, and 0.5 pM of reporter vector and 1 pM of effector vector were used for cotransfection.…”

“…Reporter vectors were pUL3(−777/+324)-Luc, pUL3(−777/+225)-Luc, pUL3(−342/+324)-Luc, and pUL3(−777/−54)-Luc. The effector vectors expressing EHV-1 regulatory proteins were pIE, pIR2, pEICP0, pIR4, pUL5, and pETIF (Ahn et al, 2007) as well as pCMV-UL3. Six micro litters of lipofectin (Invitrogen) were mixed with 100 μl of Opti-MEM medium (Gibco, BRL, Gaithersburg, MD), and incubated for 45 min at room temperature.…”

“…Early regulatory gene products include the EICP0 protein (IECP0P), IR4 protein (IR4P) and UL5 protein (UL5P) that independently or synergistically with the IEP trans -activate early and late genes (Bowles et al, 2000; Holden et al, 1995; Smith et al, 1992). In addition, three early EHV-1 gene products, the IR2 protein (IR2P) (Harty and O’Callaghan, 1991; Kim et al, 2006), UL4 protein (UL4P) (Charvat et al, 2011), and IR3 RNA (Ahn et al, 2007, 2010), play a role in regulating viral gene expression as negative regulatory molecules.…”