Kinetics of transcription of infectious laryngotracheitis virus genes

Abstract

The kinetics of expression of only a few genes of infectious laryngotracheitis virus (ILTV) have been determined, using northern blot analysis. We used quantitative reverse transcriptase PCR to examine the kinetics of expression of 74 ILTV genes in LMH cells. ICP4 was the only gene fully expressed in the presence of cycloheximide, and thus classified as immediate-early. The genes most highly expressed early in infection, and thus classified as early, included UL1 (gL), UL2, UL3, UL4, UL5, UL6, UL7, UL8, UL13, UL14, UL19, UL20, UL23 (TK), UL25, UL28, UL29, UL31, UL33, UL34, UL38, UL39, UL40, UL42, UL43, UL44 (gC), UL47, UL48 (α-TIF), UL49, UL54 (ICP27), US3 and US10. ORF A, ORF B, ORF C, ORF E, sORF 4/3, UL[−1], UL0, UL3.5, UL9, UL10 (gM), UL11, UL15a, UL15b, UL18, UL22 (gH), UL24, UL26, UL30, UL32, UL36, UL45, UL49.5 (gN), UL52, US2, US4 (gG), US5 (gJ) and US9 were most highly expressed late in infection and were thus considered late genes. Several genes, including ORF D, UL12, UL17, UL21, UL27 (gB), UL35, UL37, UL41, UL46, UL50, UL51, UL53 (gK), US8 (gE), US6 (gD) and US7 (gI), had features of both early and late genes and were classified as early/late. Our findings suggest transcription from most of ILTV genes is leaky or subject to more complex patterns of regulation than those classically described for herpesviruses. This is the first study examining global expression of ILTV genes and the data provide a basis for future investigations of the pathogenesis of infection with ILTV.

Introduction

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes severe respiratory tract disease in chickens throughout the world and is capable of causing outbreaks with a high morbidity and mortality. Its biology appears similar to that of other lytic herpesviruses [1], with a capacity to cause acute lytic infections of epithelial cells and to establish latency in neurones. Different strains of ILTV vary in virulence and their tropism for the trachea and conjunctiva [2]. Several deletion mutants of ILTV have been generated and used to examine the role of specific genes in pathogenesis, and these studies have suggested that the genes for UL0, TK, gJ and gG are not essential for infection, but do play a significant role in virulence [3], [4], [5], [6].

The ILTV genome encodes 74 predicted transcriptional units (Table 1). Apart from studies on ICP4, ORFs A–D, UL[−1], UL0, UL8, UL31, UL37, UL46–49, UL53 (gK), UL54 (ICP27) and US4 (gG) [7], [8], [9], [10], [11], there have been no detailed studies of the kinetics of expression of ILTV genes.

In lytic herpesviruses, genes are divided into immediate-early (IE), or α genes, early (E), or β genes, and late (L), or γ genes, based on their temporal expression and the dependence of their expression on other gene products. The IE gene products are mostly transcription factors and are expressed independently of de novo protein synthesis. Early genes are generally involved in viral DNA metabolism and replication. Late genes are partially or completely dependent on the replication of viral DNA and generally encode structural and other proteins involved in virion assembly. However, there are some reports indicating that the three traditional kinetic classifications of the alphaherpesvirus genes (IE, E and L) are imprecise, particularly for other herpesvirus subfamilies. Studies of temporal gene expression of human cytomegalovirus (HCMV) found five distinct kinetic groups, with E genes subdivided into β1 (early) and β2 (early late) genes, and L genes also separated into γ1 (leaky late) and γ2 (true late) genes. Differences in the regulation of maximal protein expression, changes in expression levels later in infection, and different responses to inhibitors of viral DNA synthesis separate β1 from β2 genes. Transcription of γ1 genes takes place in the presence of inhibitors of DNA replication at reduced levels, whereas transcription of γ2 genes is completely inhibited in the presence of DNA replication inhibitors. Transcription of IE genes begins 1 h after infection and peaks 4–8 h after infection, whereas transcription of β1 and β2 genes begins 4–24 h after infection. The L genes are transcribed later than 24 h after infection [12]. A study of the temporal expression of 35 genes of human herpesvirus 6B (HHV-6B) using qRT-PCR suggested the segregation of HHV-6B genes into six, instead of three, separate kinetic classes, with the subdivision of IE, E and L genes into two groups each [13].

As the kinetics of ILTV gene expression have not been fully determined, this study aimed to examine the expression of all known ILTV genes using qRT-PCR. Understanding the kinetics of ILTV gene expression may provide insights into the biology of ILTV infection and facilitate further investigations of the function of viral genes.