Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

Sokoloski, Kevin J.; Nease, Lauren M.; May, Nicholas; Gebhart, Natasha N.; Jones, Claire E.; Morrison, Thomas E.; Rw, Hardy

doi:10.1371/journal.ppat.1006473

Cited by 33 publications

(43 citation statements)

References 75 publications

(117 reference statements)

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“…Thirty minutes before the indicated times post infection, the media was removed and replaced with methionine‐ and cysteine-free DMEM (Cellgro) to starve the cells of methionine. After a 30 minute incubation period, the starvation media was removed and replaced with methionine‐ and cysteine-free DMEM supplemented with 50μM L-azidohomoalanine (L-AHA), a methionine analogue (29, 30). After 2hrs, the labeling media was removed, the cells were washed with 1x PBS, and then harvested with RIPA buffer (50mM Tris-HCl, pH 7.5 / 150mM NaCl / 1% v/v Nonidet P-40 (NP-40) / 0.5% w/v SDS / 0.05% w/v Sodium Deoxycolate / 1mM EDTA).…”

Section: Methodsmentioning

confidence: 99%

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

LaPointe

Moreno-Contreras

Sokoloski

2018

Preprint

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25Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe 26 disease and are a significant burden to public health. Alphaviral replication results in the 27 production of both capped and noncapped viral genomic RNAs, which are packaged 28 into virions during the infections of vertebrate and invertebrate cells. However, the roles 29 that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet 30 to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral 31 infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), 32 which altered the synthesis of the ncgRNAs during infection by modulating the protein's 33 capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased 34 capping efficiency, while a point mutant at D355A increased the capping efficiency of 35 the SINV genomic RNA during genuine viral infection. Viral growth kinetics were 36 significantly reduced for the D355A mutant relative to wild type infection, whereas the 37 Y286A and N376A mutants showed modest decreases in growth kinetics. Overall 38 genomic translation and nonstructural protein accumulation was found to correlate with 39 increases and decreases in capping efficiency. However, genomic, minus strand, and 40 subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral 41 capping activity. In addition, translation of the subgenomic vRNA was found to be 42 unimpacted by changes in capping efficiency. The mechanism by which decreased 43 presence of ncgRNAs reduced viral growth kinetics was through the impaired 44 production of viral particles. Collectively, these data illustrate the importance of 45 ncgRNAs to viral infection and suggests that they play in integral role in the production 46 of viral progeny. 47 94 5' termini of the alphaviral genomic and subgenomic vRNAs, which provided the first 95 evidence for independent promoter initiation for the two positive-sense vRNA species 96 (9). While these studies were able to identify the presence of the 5' type-0 cap structure, 97 they were, by the nature of their design and technological limitations, unable to 98 determine the relative frequency with which the positive sense vRNAs were capped. 99Recently, we reported findings that indicated that the alphaviral genomic vRNAs 100 are not ubiquitously capped, and that a significant proportion of the genomic vRNAs 101 produced during SINV and RRV infection lack the 5' type-0 cap structure (18). 102 Furthermore, analyses of infectious and noninfectious viral particles demonstrated that 103 both the capped and noncapped genomic vRNAs are packaged into viral particles 104 throughout the course of infection. Through the use of tissue culture models of 105 alphaviral infection, the presence of the noncapped vRNAs were found to correlate with 106 the activation of a type-I IFN response. Moreover, an attenuated RRV mutant was found 107to produce fewer noncapped genomic RNAs relative to wild type virulent RRV (18...

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Section: Methodsmentioning

confidence: 99%

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

LaPointe

Moreno-Contreras

Sokoloski

2018

Preprint

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“…Region II is a smaller stretch of amino acids (81-114) whose primary role is interaction with the RNA packaging signal (PS) [37,38]. A smaller amino acid sequence (99)(100)(101)(102)(103)(104)(105)(106)(107)(108)(109)(110)(111)(112)(113)(114) at the C-terminus of region II is highly conserved among alphaviruses. In the crystal structure, the N-terminus (regions I and II) is disordered and unresolved [25].…”

Section: The Capsid Proteinmentioning

confidence: 99%

“…There is also a line of research dedicated to understanding the role of the CP upon entry into new cells. Evidence from Sokoloski et al, 2017, suggests that specific CP-RNA interactions generated during assembly or upon disassembly can alter the kinetics of viral growth in a subsequent cell [99]. More directly related to the mechanism of assembly and perhaps the most important task is to provide reliable evidence for assembly intermediates.…”

Section: Outstanding Questions and Conclusionmentioning

confidence: 99%

Alphavirus Nucleocapsid Packaging and Assembly

Kühn

2018

Viruses

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Alphavirus nucleocapsids are assembled in the cytoplasm of infected cells from 240 copies of the capsid protein and the approximately 11 kb positive strand genomic RNA. However, the challenge of how the capsid specifically selects its RNA package and assembles around it has remained an elusive one to solve. In this review, we will summarize what is known about the alphavirus capsid protein, the packaging signal, and their roles in the mechanism of packaging and assembly. We will review the discovery of the packaging signal and how there is as much evidence for, as well as against, its requirement to specify packaging of the genomic RNA. Finally, we will compare this model with those of other viral systems including particular reference to a relatively new idea of RNA packaging based on the presence of multiple minimal packaging signals throughout the genome known as the two stage mechanism. This review will provide a basis for further investigating the fundamental ways of how RNA viruses are able to select their own cargo from the relative chaos that is the cytoplasm.

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“…Disassembly of the nucleocapsid core results in the release of the viral genomic RNA into the cytoplasmic milieu, where it may associate with the host translational machinery to initiate synthesis of the viral replicase complex. Recently, we have reported data that indicate that some viral capsid protein may remain associated with the viral genomic RNA, where it enhances early viral gene expression (20). Moreover, it should be noted that a large number of incoming genomic RNAs fail to engage with the host machinery and undergo rapid degradation following entry into the cell (20,21).…”

mentioning

confidence: 99%

Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions

LaPointe

Gebhart

Meller

et al. 2018

J Virol

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Arthropod-borne viruses, such as the members of genus , are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduces a robust viral RNA:protein discovery method to elucidate the Sindbis virus (SINV) RNA:Protein host interface. Cross-Link Assisted mRNP Purification (CLAMP) assessment reveals an extensive array of host/pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of Protein:vRNA interaction via a CLIP-seq approach and assessed the consequences of the RNA:protein binding event of hnRNP K, hnRNP I, and hnRNP M in regards to viral infection. Herein we demonstrate that mutation of the prioritized hnRNP:vRNA interaction sites effectively disrupted the hnRNP:vRNA interaction. Correlating with disrupted hnRNP:vRNA binding, SINV growth kinetics were reduced relative to wild type parental viral infections in a vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics were found to be dysregulated structural gene expression. Collectively, this study further defines the scope and importance of the alphavirus host/pathogen vRNA:protein interactions. Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their viral lifecycle. However, the extent, and importance, of host/pathogen RNA:protein interactions is woefully under characterized. The efforts detailed in this study fulfill this critical gap; and the significance of this research is three-fold. First, the data presented here fundamentally expands the scope and understanding of alphavirus host/pathogen interactions. Secondly, this study identifies the site of interactions for several prioritized interactions and defines the contribution of the RNA:protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of given host/pathogen interactions may be assessed, in the future, using a mouse model of infection.

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Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

Cited by 33 publications

References 75 publications

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

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

Alphavirus Nucleocapsid Packaging and Assembly

Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions

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