Characterization of Three Deletion Variants of Herpes Simplex Virus Type-1 (HSV-1): Sequence, Latency and Virulence Analysis

Virology

Junejo, Fazil

University:University of Glasgow

关键词: Virology;   

Others  :  http://theses.gla.ac.uk/78302/1/11008034.pdf

来源: University of Glasgow

【 摘 要 】

The aim of the work described in this thesis was to further characterise three spontaneously derived deletion variants of herpes simplex virus type-1 (HSV-1) strain 17 syn+ , designated as 1704, 1705 and 1706 (MacLean and Brown, 1987b). The characterization included, (1) sequencing across the end points of deletions by the dideoxy chain termination reaction method , to (a) investigate the relationship of the variants to each other, since they had arisen from a single recombination experiment (b) determine the extent of the deletions with respect to the location of the latency associated transcripts (LATs) and the LAT promoter region and (2) to analyse the pathogenic and latency phenotype of the three variants in the mouse model system. The characterisation , of the variants 1704, 1705 and 1706 was carried out by restriction enzyme digestion of virus DNA, selective oligonucleotide hybridisation and more precisely by sequencing across the end points of the deletion by the dideoxy chain termination reaction method. In the variant 1704, the deletion in UL/IRL is 3758bp in length, starting at nucleotide position (np) 116502 and ending at npl20260. The deletion removes 655bp of UL and 3103bp of IRL. The UL56 gene and 799bp of the 5' end of the latency associated transcripts (LATs) are deleted including the LAT promoter region. In TRL the deletion is 942bp in length extending from np7202 to 8144 and is confined entirely within TRL. The 5' end of the LAT is not affected but the LAT promoter region is deleted. Sequencing analysis of the variant 1705 showed that the deletion in UL/IRL is 4735bp in length, extending from np 115453 to np 120188. This deletion is 183bp and 694bp downstream from the 3' ends of the IE2 and IE1 genes respectively and removes the genes UL55 and UL56. One copy of the LAT coding region plus the LAT promoter region is deleted. The variant 1705 is not deleted in TRL. Sequencing analysis of the variant 1706 showed that it has a 1807bp deletion at the right hand end of UL which has been replaced by 4754bp from the left end. The deletion starts just 80bp downstream from the 3' end of the IE2 gene and terminates at the UL/IRL junction. The deletion therefore completely removes the UL55 and UL56 genes. The deleted sequences are replaced by sequences from the left end of UL containing the genes UL1, UL2, UL3, UL4 and a partial copy of UL5 in an inverted orientation. To study the biological properties of the variants, a baseline was established from which to evaluate pathogenicity. Nine individual plaques were picked from the elite stock of 17 syn+; restriction enzyme analysis of the DNA from each of the nine plaque stocks showed no differences in the size of fragments or distribution of the sites. These plaques were inoculated intracranially into three week old BALB/c mice and showed no differences in their LD50 values compared to the parental 17 syn+ stock. Inoculation of the variants, 1704, 1705 and 1706 into 3 week old BALB/c mice showed that 1705 was not different in pathogenicity from the wild type following intracranial, footpad and intraperitoneal inoculations. Therefore, despite the deletion, 1705 consistently behaved as wild type. On the other hand 1704 and 1706 compared to wild type were 20 fold and 460 fold less virulent respectively following intracranial inoculation and failed to kill any animal following footpad inoculation, even at doses of 10 pfu/mouse. In in vivo replication experiments in the peripheral nervous system (DRG of the spinal cord) of mice 1704 and 1706 grew very poorly. Latency analysis of the variants showed that the three variants established, maintained and reactivated from latency. The kinetics of reactivation of 1705 and 1706 were similar to the parent 17 syn+, in which reactivation occured 5-6 days post explantation, but 1704 reactivated with delayed kinetics i.e on the 12th day post explantation. Since 1704 has deleted both copies of the LAT promoter region and one copy of the LAT coding region in IRL, it was concluded that the LATs play a part in latency reactivation of 1704 from DRG (dorsal root ganglia of spinal cord) in the mouse model. Restoration of the deleted sequences in the variant 1704 by marker rescue with the wild type BamHI b fragment resulted in a wild type genotype. This virus was designated 1704R. Latency studies on 1704R revealed that the rate and frequency of reactivation was intermediate between 17 syn+ and 1704, suggesting a secondry undetected mutation affecting latency phenotype. Isolation of 1704LP- in which both copies of the promoter region of the LAT are deleted and reactivation of this virus from latency with delayed kinetics confirms that the LATs paly a role in reactivation from latency.