Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

ABSTRACT Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infection...

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Autores principales:	Autumn T. LaPointe, Joaquín Moreno-Contreras, Kevin J. Sokoloski
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	RNA processing RNA virus alphavirus molecular biology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9021dd70f976456b9f8b76ecdf053050
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spelling	oai:doaj.org-article:9021dd70f976456b9f8b76ecdf0530502021-11-15T15:52:18ZIncreasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection10.1128/mBio.02342-182150-7511https://doaj.org/article/9021dd70f976456b9f8b76ecdf0530502018-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02342-18https://doaj.org/toc/2150-7511ABSTRACT Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infections of vertebrate and invertebrate cells. However, the roles that the ncgRNAs play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. Specifically, point mutations at residues Y286A and N376A decreased capping efficiency whereas a point mutation at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics levels were significantly reduced for the D355A mutant relative to wild-type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation were found to correlate with increases and decreases in capping efficiency. However, genomic, minus-strand, and subgenomic viral RNA synthesis were largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic alphaviral RNA (vRNA) was found not to be impacted by changes in capping efficiency. The mechanism by which the decreased presence of ncgRNAs reduced viral growth kinetics levels operated through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggest that they play an integral role in the production of viral progeny. IMPORTANCE Alphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.Autumn T. LaPointeJoaquín Moreno-ContrerasKevin J. SokoloskiAmerican Society for MicrobiologyarticleRNA processingRNA virusalphavirusmolecular biologyMicrobiologyQR1-502ENmBio, Vol 9, Iss 6 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA processing RNA virus alphavirus molecular biology Microbiology QR1-502
spellingShingle	RNA processing RNA virus alphavirus molecular biology Microbiology QR1-502 Autumn T. LaPointe Joaquín Moreno-Contreras Kevin J. Sokoloski Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
description	ABSTRACT Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infections of vertebrate and invertebrate cells. However, the roles that the ncgRNAs play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. Specifically, point mutations at residues Y286A and N376A decreased capping efficiency whereas a point mutation at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics levels were significantly reduced for the D355A mutant relative to wild-type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation were found to correlate with increases and decreases in capping efficiency. However, genomic, minus-strand, and subgenomic viral RNA synthesis were largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic alphaviral RNA (vRNA) was found not to be impacted by changes in capping efficiency. The mechanism by which the decreased presence of ncgRNAs reduced viral growth kinetics levels operated through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggest that they play an integral role in the production of viral progeny. IMPORTANCE Alphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.
format	article
author	Autumn T. LaPointe Joaquín Moreno-Contreras Kevin J. Sokoloski
author_facet	Autumn T. LaPointe Joaquín Moreno-Contreras Kevin J. Sokoloski
author_sort	Autumn T. LaPointe
title	Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
title_short	Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
title_full	Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
title_fullStr	Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
title_full_unstemmed	Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
title_sort	increasing the capping efficiency of the sindbis virus nsp1 protein negatively affects viral infection
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/9021dd70f976456b9f8b76ecdf053050
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Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

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