Identification of the immediate-early products of bovine herpesvirus-1

Abstract

Bovine herpesvirus-1 (BHV-1) is a member of the alphaherpesvirinae. Productive infections of the alphaherpesvirinae have short infectious cycles that progress through three stages of viral transcription and translation. The first stage, immediate-early (IE), does not require prior viral gene expression and is re­ quired for the second, delayed-early (DE), and third, late (L), stages of the infectious cycle. Like other herpesviruses, BHV-1 frequently establishes latent infections. The L stage of BHV-1 infections has been well characterized at the molecular level. The DE stage has been less thoroughly investigated, while the IE stage has remained poorly characterized. The molecular biology of establish­ ment of and reexpression from the latent state is also poorly characterized.This study was undertaken to better characterize the IE stage of the productive BHV-1 infection. The number and location of IE genes and the size of IE polypeptides and IE mKNAs were investigated. Probing genomic Southern blots of BHV-1 cloned in pBK322 with ^^P-in vivo labeled cytoplasmic lE-RNA reveals six regions of hybridization located in Hindin clones D (two regions of 5.9 and 3.2 kilobase pairs [kb]), J (two regions of 2.7 and 1.5 kb), and K (two regions of 3.0 and 1.35 kb). These results suggest as many as six IE genes. Sodium dodecylsulphate-polyacrylamide gel electro­ phoresis of 35S-methionine (met)-in vivo labeled BHV-1 polypeptides reveals at least three IE polypeptides of 180,140, and 46 kilodaltons (kD). While 3%methionine (met) labeled in vitro translation of poly A+ lE-KNA reveals four polypeptides of 180,150,140, and 46 kD. 35s-met labeled hybrid arrested-in vitro translation of poly A+ lE-RNA shows that two subclones of D (pHDP6.5 and pHDP4.3) encode the three polypeptides of 180,140, and possibly, 46 kD. The polypeptide profile of hybrid arrestedin vitro translated poly A+IE-RNÂ using clones J and K to block translation were identical to unhybridized lE-RNA controls. Northern analysis of lE-RNA using pHDP4.3 as probe reveals only one transcript of 5.2 kb. These data suggest the 180 kD product is processed to the 140 and possibly the 46 kD products or that two or three polypeptides may be translated from the same mRNA. Thus, BHV-1 (Cooper) contains at least one IE gene located within HindlH fragment D. This gene is transcribed to an mRNA of 5.2 kb The 5.2 kb mRNA encodes two poly­ peptides of 180 and 140 kD, and possibly, a third of 46 kD. It was not determined if the 180 kD polypeptide is processed to yield the 140 and 46 kD polypeptides or if the 5.2 kb mRNA contains multiple sites for initiation of translation. One in vitro translation ecperiment of poly A+ lE-RNA labeled with leucine (leu) revealed seven polypeptides of 180,150,140,46,39,37, and 35 kD. This suggests that several IE polypeptides may not contain methionine. The relationship among the other Ave possible IE genes and the leu-containing polypeptides of 150,39,37, and 35 kD was not investigated.