Characterization of Novel RNA Products in HSV-1 LAT Region

• Main Authors: Ting-Chi Chen, 陳廷祺
• Other Authors: 張淑媛
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/46701627700190178933

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 96 === Human herpes simplex virus type 1 (HSV-1) is a double-strand DNA virus with about 152.0KB genome. It infects mucus epithelial cells primarily with no symptoms and establishes latent infection in trigeminal ganglia neuron cells. When host suffers from stress, HSV-1 would reactivation and cause cold sores, keratitis and rarely fatal encephalitis. The expression of HSV-1 gene is in a cascade initialed by expression of immediate-early genes (IE genes) which regulate other host and viral genes, followed by early genes (E genes) which are related to DNA synthesis and nucleotide metabolism. Finally, late genes (L genes) regulated by IE and E genes express and encode proteins which compose viral particle. During latent state, most of viral genes are inactive but there are a group of transcripts accumulated in latent-infected neuron cells, called latency-associated transcripts (LATs). According to molecular stability, the LATs can be divided into two groups: major LATs (2.0Kb, 1.5Kb and 1.45Kb) and minor LATs (8.3Kb and 6.3Kb). The most abundant stable LAT is a 2.0Kb LAT. 2.0Kb LAT is an intron spliced from a primary minor 8.3Kb LAT. 2.0Kb LAT expresses as viral late phase gene during lytic cycle and accumulates abundantly while virus established latent state. In previous studies, we knew the 2.0Kb LAT is correlated with establishment and maintenance of viral latency, reactivation from latent state, neuron survival and antiapoptosis. In study of 1.5Kb splicing, we used reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We discovered two novel products called R200 (216bp) and R300 (306bp). Further, we found R300 may come from 2.0Kb RNA. The expression pattern of R200 and R300 is the same with L gene and suggested that R200 and R300 should express at late phase in viral lytic cycle. It has been reported that 2.0Kb LAT might influence an IE gene, ICP0, expression by blocking its splicing and this might lead virus into latent phase. We proposed that R300 and R200 which are similar to LAT may influence viral replication and promote virus into latent state. We expressed two fragments in Vero cells and infected with HSV-1. We discovered there were no difference between transfercted and wild type Vero cells and suggested that R300 and R200 have no effect on viral replication. Besides we used neuron cell line, SH-SY5Y, as target and can detect R300 and R200. We also used different primers for PCR to find the 3’ and 5’ end of RNA contained R300 and R200. We suggested that 3’ end of them should be up to LATF, but 5’ end of them didn’t include the region of LATR to LATR2. According to the sequence and RNA structure, we supposed that the mechanism of R300 splicing is group I intronic splicing. Howerer, we couldn’t predict the possible one of R200. Finally, we predicted a microRNA in 1.5Kb LAT splicing region, and its possible human gene is NFIX.