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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 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.

SUBMITTER: LaPointe AT

PROVIDER: S-EPMC6299483 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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

LaPointe Autumn T AT Moreno-Contreras Joaquín J Sokoloski Kevin J KJ

mBio 20181211 6

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 infecti ...[more]

PMID: 30538185

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Project description:Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes. IMPORTANCE Alphaviruses are important causes of viral encephalomyelitis without approved treatments or vaccines. Antibody to the Sindbis virus (SINV) E2 glycoprotein is required for immune-mediated noncytolytic virus clearance from neurons. We used direct RNA nanopore sequencing to evaluate how anti-E2 antibody affects SINV replication at the RNA level. Antibody altered the viral RNAs produced by decreasing the proportion of subgenomic relative to genomic RNA and suppressing production of a previously unrecognized defective viral genome (DVG) encoding nsP1, the viral RNA capping enzyme. Antibody-treated neurons released a lower proportion of SINV particles with capped genomes necessary for translation and infection. Decreased nsP1 DVG production in antibody-treated neurons led to lower expression of nsP1 protein, decreased genome capping efficiency, and release of fewer infectious virus particles. Capping was increased with exogenous expression of the nsP1 DVG. These studies identify a novel alphavirus DVG function and new mechanism for antibody-mediated control of virus replication.

Project description:Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.

Dataset Information

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

Publications

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

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