VIROLOGY 191, 988-991 (1992) The Latency-Related Gene of Bovine Herpesvirus 1 Inhibits the Activity of Immediate-Early Transcription Unit 1 ANA C. BRATANICH, NANCY D. HANSON, AND CLINTON J. JONES’ University of Nebraska, Lincoln, Center for Biotechnology, Department of Veterinary Sciences, Fair Street at East Campus Loop, Lincoln, Nebraska 68583-0905 Received June 15, 1992; accepted September 2, 1992 Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory neurons of infected animals. Only one virus-en- coded latency-related (LR) gene is expressed during a latent infection. The LR transcript overlaps immediate-early transcription unit 1 (IEtul) and is anti-sense with respect to IEtul. The transcriptional start site of the LR RNA was mapped to position 724 of the LR gene, downstream from two putative TATA elements. When LR promoter sequences were deleted from a plasmid containing IEtul and the LR gene, the resulting construct trans-activated the HSV-1 thymidine kinase (TK) promoter more efficiently than IEtul plus the LR gene. Cotransfection of a plasmid containing the intact LR gene with IEtul inhibited the ability of IEtul to trans-activate the TK promoter. These results imply that a LR gene product(s) repressed the trans-acting capacity of IEtul. o 1992 Academic press, hc. BHV-1 is a member of the alphaherpesvirus family and is an important viral pathogen of cattle. Infection can lead to a variety of syndromes in the respiratory tract, nervous system, or the genital tract (reviewed in 19). Like all alphaherpesviruses, a latent infection is established in the peripheral nervous system (16, 19). During a latent infection, a single latency related (LR) transcript is detected in the nucleus of ganglia (7, lo- 13). This transcript maps to a region of the viral ge- nome which overlaps immediate-early transcription unit 1 (IEtul; 7, 17). The LR transcript overlaps IE2.9 as well as an early transcript (E2.6) and is transcribed from the opposite strand of DNA with respect to IE2.9 and E2.6. It is not clear what role, if any, the LR gene plays during a latent infection. During reactivation of latently infected animals, levels of LR transcripts decrease (13), suggesting that expression of the LR gene plays a role during some phase of the latent infection. Studies with HSV-1 have suggested that the latency-associated transcript is involved with reactivation (5, 8) or estab- lishment of latent infections (14). It is also clear that cellular factors in neurons play a crucial role with re- spect to repressing immediate early (IE) gene expres- sion (3, references therein). Apparently cellular as well as viral factors are necessary for establishing and maintaining a latent infection. In this study, the start site of transcription for the LR gene of BHV-1 was mapped to sequences which are adjacent to two overlapping TATA boxes within the ’ To whom reprint requests should be addressed. promoter. This information was used to construct a plasmid which contained IEtul but not the LR pro- moter/regulatory sequences (IEtu l/ALR). The trans- acting capacity of IEtul/ALR was compared to a plas- mid which contains IEtul plus the intact LR gene. These studies revealed that removal of LR promoter sequences enhanced the ability of IEtul to trans-acti- vate the HSV-1 TK promoter. Addition of a plasmid containing the LR gene repressed IEtul trans-acting potential confirming the hypothesis that a LR prod- uct(s) had a negative effect on IEtul activity. To determine the transcriptional start site of the LR RNA, bovine turbinate (BT) cells were infected with BHV-1 and primer extension analysis was performed using total RNA prepared from infected cells. In several independent experiments an extended product of 117 nt was detected at 18 hr postinfection (p.i.), but not in mock-infected cells (Fig. 1). Prolonged exposure of the gel revealed that this 117-nt extended product was also present in cells infected for 4 hr but not in mock- infected cells. In some experiments an extended prod- uct of 120 nt was present, suggesting that multiple start sites exist for LR transcription. The band larger than 120 nt was always present in mock-infected cells to varying degrees and thus was not virus specific. The transcription initiation site of the 1 17-nt product corre- sponds to nt 724 of the LR gene (7) which is adjacent to two putative TATA boxes (Fig. 2). A statistical study on TATA boxes concluded that in general, the start site of transcription begins 24-36 nt from the most 5’T of a TATA box (9). The LR initiation site is 24 or 25 nt from the Ts at nt 699 or 700 in the LR gene. In the same statistical study, A is the first transcribed nucleotide in 0042.6822/92 $5.00 988 Copyrtght 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.