Isolation and localization of herpes simplex virus type 1 mRNA

Abstract

Herpes simplex virus (HSV) DNA bound to cellulose has been 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 has sequences encoded by approximately 45% of the HSV DNA. This RNA has a size range of from 1.5 to-8 kilobases, with certain sizes, such as 1.7 to 1.9 kilobases, being favored. We have used the restriction endonucleases HindIII and XbaI singly and together to generate various sized fragments covering the entire HSV-1 genome. These fragments have been 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. We used hybridization of size-fractionated RNA to Southern blots of restriction fragments of HSV-1 DNA generated by the BglII as well as HindIII and XbaI endonucleases 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 two areas of the long unique region of the HSV-1 genome which annealed to a large number of HSV-1 transcripts. Furthermore, discrete-sized mRNA species larger than 5 kilobases in length were found only in the middle of the long unique region. The implications of these data are discussed.

High-resolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein

“…Then cells were preincubated for 1 h and treated for 5 h with 1.5 x 1i-' M adenosine arabinoside and 3.7 x 106 M pentostatin to inhibit viral DNA replication, as described previously (15). RNA was size fractionated by electrophoresis on 1.4% agarose gels containing 10 mM methylmercury hydroxide (3) as previously described (1,2).…”

“…Restriction fragment-specific mRNA was isolated from HSV poly(A) mRNA by preparative hybridization to the appropriate DNA covalently coupled to cellulose. Details of coupling of DNA to cellulose and preparative hybridization were as described previously (2,6).…”

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome

“…Rinsing of cellulose and elution of hybridized RNA were as described'previously (5). In the experiments described here, samples of 50 to 60 pg of 3P-labeled late poly(A) polyribosomal mRNA were hybridized with 100-pig equivalents of a given restriction fragment for 4 h (a microgram equivalent is that amount of a specific fragment obtainable from 1 pg of HSV-1 DNA).…”

“…After viral DNA replication, another large group of viral genes is expressed. These are the late or y genes (4,5,16,18,19,21,36;Wagner et al., in press). A large number of these code for viron structural proteins.…”

An unusual spliced herpes simplex virus type 1 transcript with sequence homology to Epstein-Barr virus DNA

“…This is referred to as HSV poly(A) mRNA. Details of this procedure have been presented elsewhere (1,2,(10)(11)(12)(13)(14)16). RNA was isolated at 6 h postinfection.…”