BMB-10 Impact of SARS-CoV NSP1 on mRNA Transcription and Metabolism
Abstract
Introduction: Nonstructural Protein 1 (NSP1) of severe acute respiratory syndrome coronavirus (SARS-CoV) selectively inhibits host mRNA translation by blocking the translation initiation site of the 40s ribosomal subunit and inducing endonucleolytic cleavage of host mRNAs while allowing viral RNA to translate. Recent research in our laboratory suggests NSP1 may also impact mRNA transcription in the nucleus and RNA metabolism in the cytoplasmic stress granule. Despite these preliminary findings, the exact mechanism of NSP1’s function in the nucleus and cytoplasm remains unknown.
Goal of study: To investigate how NSP1 impacts host mRNA synthesis in the nucleus, specifically how it alters mRNA transcription and 3’-end processing, we seek to identify nuclear proteins that interact with NSP1 and alter pre-mRNA splicing and polyadenylation. To investigate how NSP1 impacts RNA localization in the stress granule, we seek to study the localization of host mRNAs and representative viral transcript.
Methods and results: To identify nuclear interactors of NSP1, purified nsp1 was used to pull down specific proteins from nuclear extract, separated using the SDS page, and were further visualized using silver stain. Strong interactors will be identified using mass spectrometry. In addition, we are conducting RNA-sequencing of nuclear mRNAs in the presence and absence of NSP1 to validate NSP1’s role in modulating pre-mRNA splicing and polyadenylation. We plan to systematically optimize the tagging and labeling of host and viral RNA in stress granules in the presence of NSP1 and monitor their localization at different time points after inducing stress.
Conclusion: We have currently established that NSP1 interacts with various host cell nuclear proteins, although the specific proteins themselves have yet to be determined. We have eliminated certain methods for the visualization of the viral RNA and are currently working on optimizing our fluorescent in situ hybridization technique to best visualize both host and viral RNAs.
Grant support: This research was partially supported by the Developmental Research Project Program by SC INBRE (National Institutes of Health, National Institute of General Medical Sciences, P20GM103499) and the R15 grant (National Institute of Allergy and Infectious Diseases).
Keywords
SARS-CoV, Covid-19, Non structural protein 1
BMB-10 Impact of SARS-CoV NSP1 on mRNA Transcription and Metabolism
University Readiness Center Greatroom
Introduction: Nonstructural Protein 1 (NSP1) of severe acute respiratory syndrome coronavirus (SARS-CoV) selectively inhibits host mRNA translation by blocking the translation initiation site of the 40s ribosomal subunit and inducing endonucleolytic cleavage of host mRNAs while allowing viral RNA to translate. Recent research in our laboratory suggests NSP1 may also impact mRNA transcription in the nucleus and RNA metabolism in the cytoplasmic stress granule. Despite these preliminary findings, the exact mechanism of NSP1’s function in the nucleus and cytoplasm remains unknown.
Goal of study: To investigate how NSP1 impacts host mRNA synthesis in the nucleus, specifically how it alters mRNA transcription and 3’-end processing, we seek to identify nuclear proteins that interact with NSP1 and alter pre-mRNA splicing and polyadenylation. To investigate how NSP1 impacts RNA localization in the stress granule, we seek to study the localization of host mRNAs and representative viral transcript.
Methods and results: To identify nuclear interactors of NSP1, purified nsp1 was used to pull down specific proteins from nuclear extract, separated using the SDS page, and were further visualized using silver stain. Strong interactors will be identified using mass spectrometry. In addition, we are conducting RNA-sequencing of nuclear mRNAs in the presence and absence of NSP1 to validate NSP1’s role in modulating pre-mRNA splicing and polyadenylation. We plan to systematically optimize the tagging and labeling of host and viral RNA in stress granules in the presence of NSP1 and monitor their localization at different time points after inducing stress.
Conclusion: We have currently established that NSP1 interacts with various host cell nuclear proteins, although the specific proteins themselves have yet to be determined. We have eliminated certain methods for the visualization of the viral RNA and are currently working on optimizing our fluorescent in situ hybridization technique to best visualize both host and viral RNAs.
Grant support: This research was partially supported by the Developmental Research Project Program by SC INBRE (National Institutes of Health, National Institute of General Medical Sciences, P20GM103499) and the R15 grant (National Institute of Allergy and Infectious Diseases).