The molecular mechanism of the role of human cytomegalovirus protein pUL38 employing autophagy in regulating ER stress induced cell death

I.Summary

Viral infection triggers cellular stress responses.For instance,endoplasmic

reticulum stress(ER).Growing evidence implicates ER stress induces autophagy through the suppression of mTORC1.In addition,Z.K.Qian et al reported that the human cytomegalovirus protein pUL38 can modulate ER stress by limiting premature cell death and maintaining mTORC1 activity individually during viral infection.As a highly conserved process from yeast to mammals,autophagy plays a controversial role in regulating cell death and survival..It remains elusive how pUL38 modulating ER stress is related to autophagy regulation.Here I propose strategies to unravel the mechanism of HCMV protein pUL38 regulates ER stress induced autophagy by combining molecular cell biological,biochemical,and imaging tools.Understanding the mechanism of pUL38 modulation process will expand our knowledge on the interface of virus-host interactions,and will be helpful for drug development against globally pathogens like HCMV,which may be beneficial for immuno-compromised patients,such as cancer patients,patients with HIV infection,various vascular diseases,and recipients of bone marrow and solid organ transplants.

II.Specific Aims

1.      ER stress triggers autophagy-mediated cell death during pUL38-deficient virus infection.

2.      ER stress induces autophagy through the suppression of mTORC1 during pUL38-deficient virus infection.

III.Introduction

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IV.Experimental Plan

Specific Aim1:ER stress triggers autophagy-mediated cell death during pUL38 deficient virus infection

      Previous study reported that HCMV pUL38-deficient virus induced premature cell death.Cell death was induced in HF cells either by infection of pUL38-deficient HCMV or by treatment of cells with the ER stress-inducing agent tunicamycin.Excessive ER stress leads to cell death.Remarkable cell death will be observed via microscope and MTT assay.Therefore,I examined the cleaved form of caspase-3 or PS markers(label phosphatidylserine translocation)together with microscopic analysis for cell morphology and the conversion of LC3 which respectively indicate the occurrence of apoptosis and autophagy.Moreover,in order to exclude the possibility that the LC3 conversion was due to the autophagosome-lysosome fusion,we could block the vacuolar fusion with commercial inhibitors.Taking advantage of drug stimulation,we will speculate that autophagy may mediate ER stress-induced cell death during pUL38 deficient HCMV infection.

Specific Aims2:ER stress induces autophagy through the suppression of mTOR during pUL38 deficient virus infection.

      Increasing evidence suggest laying a bidirectional regulation pattern between autophagy and ER stress.However,the role of mTORC1 in ER stress induced autophagy is still an open question.I will employ pUL38-deficient HCMV infection model to induce ER stress,and determine mTORC1 activity by labeling p-S6 and 4E-BP1,compared with wildtype HF cells.To provide additional evidence that the inhibitory activity of mTORC1 in ER stress-induced autophagy, we will treat TSC-deficient HF cells with pUL38 deficient HCMV virus to check autophagy conversion in comparison with their wt control cells.

V.Significance to HCMV biology and pathogenesis research

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VI.Training and background

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VII.Reference

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