Identification of gene products encoded by the latency-related gene of bovine herpesvirus 1

Abstract

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of infected animals. Expression of latency-related (LR) gene products is controlled by a 980-bp fragment (LR promoter).DNA sequence analysis revealed that two major open reading frames (ORFs) are in the LR gene. Antibodies directed against both ORFs were generated in rabbits by using synthetic peptides. Antibody P2, which is directed to sequences near the amino terminus of ORF 2, recognized a 41-kDa protein in lytically infected cells, suggesting that ORF 2 encodes a protein. When the LR gene was inserted into a mammalian expression vector and subsequently transfected into COS-7 cells, a 41-kDa protein was detected by use of silver-stained sodium dodecyl sulfate-polyacrylamide gels and by the P2 antibody. In contrast, this protein was not detected in mock-transfected cells. Deletion of DNA sequences containing ORF 2 blocked synthesis of the 41-kDa protein in COS-7 cells. Reverse transcriptase-mediated PCRs indicated that splicing occurs near the C terminus of ORF 2. Further studies indicated that LR RNA was alternatively spliced in latently infected cattle and that a fraction of LR RNA was poly(A) ؉ . Taken together, these studies suggested that a spliced LR transcript has the potential to encode a 41-kDa protein.

Persistence and Reactivation of Bovine Herpesvirus 1 in the Tonsils of Latently Infected Calves

“…Double-stranded DNA probes specific for BHV-1 were synthesized and labeled by PCR with digoxigenin-dUTP (Boehringer Mannheim Corp., Indianapolis, Ind.). PCR conditions using ICP4, RR, and gC upstream and downstream primers have been previously described and are listed below (12,30). The coding sequence for the BHV-1 gC glycoprotein was contained in plasmid pKS92-2 and was obtained from S. I. Chowdhury (Kansas State University).…”

Identification of a novel protein encoded by the latency-related gene of bovine herpesvirus 1

“…For this study, we designed a polyclonal antiserum directed against the majority of ORF-1 coding sequences. Although a previous study did not detect ORF-1 using a peptide antibody, the ORF-1 peptide antibody was directed to sequences near the Nterminus (Hossain et al, 1995). Because LR-RNA undergoes splicing at or near this region (Devireddy and Jones, 1998) (Figure 1C), the peptide antiserum could not have detected an ORF-1 protein that was translated from a spliced transcript.…”

“…Consequently, the LR mutant virus does not express detectable levels of ORF-2, RF-C, or their respective fusion proteins that would normally be expressed as a result of alternative splicing (Jiang et al, 2004). Antiserum directed against a peptide near the N-terminus of ORF-1 ( Figure 1C) did not reproducibly recognize a viral-specific protein in infected or transfected cells (Hossain et al, 1995). The major spliced LR-RNA detected during productive infection or in TG at 7 days after infection lacks sequences encompassing the N-terminus of ORF-1 because of splicing (Devireddy et al, 2003) ( Figure 1C).…”

“…(A) Partial restriction map, location of LR-RNA, organization of LR ORF, and 3 terminus of the bICP0 ORF. The start sites for LR transcription during latency and productive infection were previously described (Devireddy and Jones, 1998;Hossain et al, 1995). Reading frames B and C (RF-B and RF-C) each contain an open reading frame that lacks an initiating Met.…”

“…The latency-related (LR) RNA is abundantly expressed in latently infected neurons (Kutish et al, 1990;Rock et al, 1987Rock et al, , 1992. A fraction of LR-RNA is polyadenylated and alternatively spliced in TG, suggesting this RNA encodes a family of proteins (Devireddy and Jones, 1998;Hossain et al, 1995). The LR gene contains two open reading frames (ORF-1 and ORF-2) and two reading frames that lack initiating ATGs (reading frame [RF]-B and RF-C) (see Figure 1A) (Kutish et al, 1990).…”