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Title: Analysis of the LAT promoter of herpes simplex virus type 1
Author: Morrow, John Anthony
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
A major feature of the biology of herpes simplex virus type 1 (HSV-1) is it's capacity to establish a latent infection in peripheral nervous system ganglionic neurons and to periodically reactivate to produce recrudescent disease. Until recently the extent and the nature of any expression from latent HSV genomes was unknown. In 1987, latency associated transcripts (LATs) were detected in murine ganglia which were latently infected with HSV-1. The LATs map to the long repeat region and are transcribed in the opposite direction to and partially overlap with IE-1 mRNA. Subsequent work has refined our knowledge of the HSV-1 LATs and shown that transcripts associated with latency are also encoded by a number of other herpesviruses including pseudorabies virus in swine and bovine herpes virus in rabbits. It is now known that the HSV-1 LATs comprise at least 3 overlapping non-polyadenylated RNAs which share common 5' and 3' ends, but differ in size through differential splicing. These LAT species are thought to be stable introns or stable processing products of a less stable 8.3 kb (m-LAT) polyadenylated primary transcript. Since LAT appears to be the only actively transcribed gene during latency, the control of LAT gene expression must differ considerably from that of any of the lytic cycle genes. A great deal of attention has thus been focussed on the LAT promoter in order to identify cis-acting sequences responsible for this unique pattern of transcription. In this study, in order to determine the location of the LAT promoter, various sequences upstream from the 2 kb LATs were cloned and examined for their ability to drive expression of an adjoining reporter gene in transient expression assays in a plasmid expression vector. The response of these sequences to the presence of HSV-1 trans- acting factors was also examined by infecting cells transfected with the various LAT promoter/CAT fusion constructs. Both the promoter and HSV-1 responsive elements mapped to a 137 bp region 800 bp upstream from the 5' end of the 2kb LATs. Sequence analysis of this region demonstrated that it contained several homologies to recognised promoter elememts including a TATA box, a CAAT box and two putative Sp1 binding sites. Comparison of this region to the corresponding region of the HSV-2 genome revealed that while the 2kb LAT transcription unit itself is completely unconserved, the putative LAT promoter region bears several interesting conserved loci, possibly indicative of the presence of enhancer like elements responsible for tissue-specific expression of the LAT gene. Several of the LAT promoter/CAT fusion constructs were examined in the context of the virus genome by use of a virus vector whereby DNA sequences of interest are introduced into the viral genome by direct ligation. To facilitate this analysis, the parental vector was modified by deletion of the endogenous LAT sequences, thereby preventing possible rearrangments of the viral genome by homologous recombination between the endogenous and introduced LAT promoter sequences. These recombinant viruses were subsequently used for analysis of the LAT promoter during infection of tissue culture cell lines. The most significant result to come from these studies was the finding that sequences upstream from the core LAT promoter, while not having any effect on LAT promoter strength in BHK cells, considerably enhanced LAT promoter strength in C1300 neuroblastoma cells, thereby suggesting that these sequences harbour neuronal specific enhancer elements. In addition to studying the LAT promoter, it was considered interesting to examine the behaviour of an endogenous neuronal specific promoter when introduced into the virus genome. To this end, the 5' flanking region of the human neurofilament-light chain gene was obtained, and two constructs, extending from -296 to +84 and -896 to +84 of the HNF-L promoter fused to the CAT reporter gene were made. Transient transfection assays showed that these constructs performed at least as well as the HSV-1 gD promoter, and were similarly strongly trans-activated upon infection of transfected cells with HSV-1. Introduction of the HNF-L/CAT fusions into the HSV-1 genome, and subsequent examination of these constructs during infection of tissue culture cell lines revealed that sequences upstream from -296, while only moderately enhancing promoter activity in BHK cells, had a greater effect in C1300 neuroblastoma cells, thereby suggesting that these sequences perhaps also containied neuronal specific enhancer elements. The original aim of these studies was to examine promoter/reporter gene activity of the various recombinant viruses in animal latency systems. However, due to lack of time and the necessity of replacing the CAT with the lacZ reporter gene for reasons of sensitivity, these studies were unable to be carried out. One virus which was available, (vFJ12) containing the HSV-2 IE-4/5 promoter fused to the lad reporter gene was used in animal latency studies. In situ analysis showed that the parental virus vector expressed LAT in latently infected murine sensory ganglia, thereby demonstrating that this virus could establish latency in this system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796785  DOI: Not available
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