PC-04 The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability
Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV1 or SARS-CoV2) encoded nonstructural protein 1 (nsp1) downregulates host gene expression by selectively stalling host mRNA translation and triggering mRNA decay.
Surprisingly, capped and polyadenylated viral RNA that closely resembles host mRNA continues to translate viral proteins. We found that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNAs that form in response to viral infection. SGs serve to protect translationally stalled mRNAs from degradation.
Since the presence of nsp1 leads to the dispersion of SG-associated proteins, we believe SGs serve as the key modulator of RNA stability in the presence of nsp1.Since viral mRNAs undergo translation in the presence of nsp1 while the translation of host mRNA is inhibited, we sought to analyze the SG localization of host mRNAs and reporter RNA carrying viral RNA sequences.
To study the RNA composition of SGs in the presence of nsp1, we transfected HEK293 cells with DNA expressing wild-type SARS-CoV nsp1. Cells were subjected to oxidative stress using sodium arsenite to mimic viral infection. SG-associated RNA was analyzed by quantitative RT-PCR to quantify mRNA localization in the presence of nsp1. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection.
When nsp1 was expressed, we found most mRNAs show a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of host mRNAs from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1.
PC-04 The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability
Severe acute respiratory syndrome coronavirus (SARS-CoV1 or SARS-CoV2) encoded nonstructural protein 1 (nsp1) downregulates host gene expression by selectively stalling host mRNA translation and triggering mRNA decay.
Surprisingly, capped and polyadenylated viral RNA that closely resembles host mRNA continues to translate viral proteins. We found that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNAs that form in response to viral infection. SGs serve to protect translationally stalled mRNAs from degradation.
Since the presence of nsp1 leads to the dispersion of SG-associated proteins, we believe SGs serve as the key modulator of RNA stability in the presence of nsp1.Since viral mRNAs undergo translation in the presence of nsp1 while the translation of host mRNA is inhibited, we sought to analyze the SG localization of host mRNAs and reporter RNA carrying viral RNA sequences.
To study the RNA composition of SGs in the presence of nsp1, we transfected HEK293 cells with DNA expressing wild-type SARS-CoV nsp1. Cells were subjected to oxidative stress using sodium arsenite to mimic viral infection. SG-associated RNA was analyzed by quantitative RT-PCR to quantify mRNA localization in the presence of nsp1. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection.
When nsp1 was expressed, we found most mRNAs show a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of host mRNAs from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1.