Isolation and localization of herpes simplex virus type 1 mRNA

Abstract

Herpes simplex virus (HSV) DNA bound to cellulose was used as a reagent to isolate viral mRNA for size analysis on denaturing agarose gels. Total viral polysomal polyadenylated RNA was isolated from cells late after infection when such RNA had sequences encoded by approximately 45% of the HSV DNA. This RNA has a size range of from 1.5 .gtoreq. 8 kilobases [kb] with certain sizes, such as 1.7-1.9 kb, being favored. The restriction endonucleases HindIII and XbaI were used singly and together to generate various sized fragments covering the entire HSV-1 genome. These fragments were bound to cellulose to allow isolation of HSV-1 mRNA annealing to different regions of the viral genome. Discrete sizes of viral mRNA are associated with certain regions of the genome, but the mRNA population hybridizing to even the smallest restriction fragments is complex. Hybridization of size-fractionated RNA to Southern blots of restriction fragments of HSV-1 DNA generated by the BglII and HindIII and Xbal endonucleases were used to confirm the preparative hybridization data and to provide some overlap data for positioning transcripts. The data of blot and preparative hybridization agreed very well and were combined to construct a preliminary transcription map of HSV-1. Such a map revealed at least 2 areas of the long unique region of the HSV-1 genome which annealed to a large number of HSV-1 transcripts. Discrete-sized mRNA species larger than 5 kb in length were found only in the middle of the long unique region. The implications of these data are discussed.