Ⅰ型疱疹病毒毒力蛋白ICP34.5与细胞自噬的相关性研究

Ⅰ型疱疹病毒毒力蛋白ICP34.5与细胞自噬的相关性研究 摘要 摘要

自噬是一种广泛保守存在于真核细胞内，作为胞内维持内环境稳态的一种 保护性机制，是细胞利用溶酶体系统把自身损坏或衰老的细胞器降解，同时也 降解外源入侵的微生物，比如病毒。在病毒入侵以后，双链RNA可以激活干扰 素诱导的抗病毒分子PKR，从而诱导白噬。PKR会将真核翻译起始因子eIF2a磷 酸化，造成整个细胞的蛋白合成的停止，从而起到抑制病毒复制的目的，这也 是干扰素防御病毒的重要作用方式之一。HSV-1(单纯疱疹病毒1型)的神经毒 力蛋白ICP34．5(由Y】34．5基因编码)在病毒的致病性和抵御宿主的抗病毒机制 中起了非常重要的作用。研究发现HSV-1的ICP34．5能够和细胞内的蛋白磷酸酶1 (PPl)结合，然后PPl可以使磷酸化的eIF20t脱磷酸化，使蛋白合成重新开始。 ICP34．5也能和自噬关键蛋EtBeclinl结合，抑制其诱导的自噬。病毒核酸被自噬 体转运至溶酶体系统过程中表达Toll样受体7，能诱导I型干扰素的产生。 本论文的研究中，我们通过病毒感染三种不同来源的细胞系，用Westernblot 的方法发现在病毒感染的一段时间以内，与缺失了Y。34．5的缺陷型病毒R3616 相比，野生型HSV-1病毒的感染能使自噬相关蛋ALc3 II的表达量增加。同时转 染实验也发现了外源转入的ICP34．5蛋白能使自噬相关蛋白LC3 II的表达量增 加。 在进一步研究中，利用Overlap PCR的方法成功构建了不与自噬关键蛋白 Beclinl结合的ICP34．5突变蛋白，并装入到真核表达载体中。我们发现ICP34．5所 诱导的LC3 II的表达含量的增高不完全依赖于与Beclinl的结合，同时它和 ICP34．5与PPl以及eIF20t的结合也是相关的。 本研究表NICP34．5在HSV-1病毒感染过程中，可以引起宿主细胞的LC3 II的 表达含量增高，即细胞自噬，这对于我们研究宿主与病毒相互作用的机制提供 了新的线索。

关键词：HSV一1，y。34．5，LC3 II，细胞白噬 Abstract Abstract

Autophagy is a conserved pathway that functions in all eukaryotic organisms to

maintain cellular homeostasis．The autophagy pathway targets for lysosomal degradation

both cellular cytoplasmic constituents，such as damaged or aged organelles，proteins

and protein aggregates，as well as microbial invaders，including viruses．After virus

infection，dsRNA structures activate the interferon inducible

antiviral molecule，PKR，which induces autophagy．PKR can phosphorylate the translation

initiation factor elF2alpha and then stop the protein synthesis and thus inhibit the virus

replication．This is one of the roles that interferon defend the

virus

invasion．The

neurovirulence protein 71 34．5 ofherpes simplex virus type I(HSV-1) plays a very

important role in the pathogenicity of the virus and in host defense

against viruses．The ICP34．5 can bind the protein phosphotase 1(PP 1)and

dephosphorylate the phorsphorylated elF2alpha to restart the protein synthesis． ICP34．5 also antagonizes the autophagy protein Beclin 1 while viral Bcl-2 family

members

antagonize Beclin 1 to suppress autophagy induction．Viral nucleic acids are

delivered

by autophagosomes to endosomes containing toll like receptor(TLR7)， which induces

type 1 interferon(IFN)production．

In our experiments，we infected three different source cell lines

by HSV-1 and

detect autophagy related protein LC3 II by Western blot．Compared with the infection of ICP34．5-deficient virus(R361 6)，wild-type

HSV-1 carl increase self-expression of

autophagy related protein LC3II during a short period．Transfection experiments of exogenous ICP34．5 also proved the same result．

Then we cloned the ICP34．5 deletion mutant which missing the binding motif with Beclin l into expression vector through the Overlap PCR．In the further study of ICP34．5 and autophagy,we found that the increased of LC3II expression which induced

by

ICP34．5 is not entirely dependent on the combination with the Beclinl，

but affected by binding PP 1 and elF2a． We claim that ICP34．5 can induce the increased of LC3II expression during HSV二

1 infection．It will be useful to study the mechanism involved in the host

II Abstract autophagy-virus interaction in our future research．

Key words：HSV-1，Y134．5，LC3

II，Autophagy

III 缩略词表 缩略词表

HSV二1 Herpes Simplex Virus

一1

ICP34．5 Infected Cell Protein 34．5

dsRNA Double stranded RNA LC3 Microtubule-associated protein 1 light chain 3 PKR dsRNA dependent protein kinase PPl Protein phosphatasel

elF2a eukaryotic

initiation factor-2

alpha

GADD34 Growth arrest and DNA damage protein 34

IFN interferon DMEM Dulbecco’S

Modified

Eagle’S medium

RPMI Roswell

Park Memorial Institute

PEI Poly

ethyleneimine

FBS fetal bovine

serum

NCS Newborn calf serum

VI