第一型單純疱疹病毒潛伏相關轉錄體區域之新RNA產物之定性

Abstract

人類單純疱疹病毒第一型(HSV-1)為線狀雙股DNA病毒，基因體大小約為152.0KB。HSV-1在初次感染時症狀不明顯，但會潛伏於神經細胞中。待宿主遭受壓力或免疫力下降，病毒會再活化，造成疱疹甚至會引起較嚴重的疾病，例如角膜炎或腦膜炎。HSV-1的基因主要分為三種。立即早期基因(Intermediate Early；IE)在病毒感染後不需要有病毒蛋白的存在即可表現，主要功用是調節細胞和病毒基因的表現。早期基因(Early；E)必須在有IE基因產生之蛋白的存在下才能表現，主要與DNA合成和核酸代謝有關。晚期基因(Late；L)受到IE及E基因的調控，主要合成病毒顆粒的組成成分。在HSV-1潛伏時，大部分HSV-1的基因表現會停止。此時唯一會大量表現的是一群RNA分子，稱為latency-associated transcripts(LAT)。LAT依照其分子穩定度可以分為major LAT與minor LAT。Major LAT較穩定，會於細胞中大量累積，其包含三種大小的分子：2.0KB、1.5Kb與1.45Kb；minor LAT有兩種：8.3Kb與6.3Kb。其中8.3Kb會經過splicing產生6.3Kb 與2.0Kb LAT。目前已知2.0KB LAT可能與HSV-1潛伏的建立與維持、病毒的再活化與抑制神經細胞凋亡有關。在1.5Kb LAT splicing site的附近，本實驗室利用oligo dT RT-PCR與sequencing的方式發現到有不同於原先研究的RNA片段的存在R300(306bp)與R200(216bp)。接著利用306bp作為probe作Northern blot確認其來源RNA大小約為2.0KB左右。同時發現其表現與L基因表現模式相似，推測是在病毒複製晚期表現。有研究報告指出2.0KB LAT可能透過影響ICP0的splicing來調控病毒的潛伏或活化。因此我們推測此和LAT有相似序列的R300與R200片段可能藉由影響病毒基因的表現，而使病毒進入潛伏狀態。於是將R200和R300分別接入CMV promoter drived質體，將其轉染至Vero細胞後感染HSV-1。發現對於HSV-1的三個時期的基因之表現或病毒的產生均無影響。此外病毒感染神經母細胞瘤細胞株SH-SY5Y，也可偵測到R200與R300的表現。接著利用不同的引子進行不同的PCR反應，推測含有R200與R300之RNA 5端應包含至LATF，3端則不含LATR至LATR2的區域。另外根據序列與二級結構預測R300的splicing過程可能為group I intron機制，但R200的splicing過程尚無法推測。最後2006年有研究指出LAT中含有miRNA，所以我們預測在1.5Kb LAT spliced區域有一可能的microRNA存在，並預測其可能的目標基因為NFIX。

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.