INTRODUCTION AND LITERATURE REVIEW
Reassortment is an important process that enhances genetic diversity and
- Reassortment occurs for all viruses with segmented genomes
- Reassortment has consequences for progeny viruses
- Segment mismatch influences reassortment
- Viruses of the Reoviridae family have segmented genomes and are
The reassortment event may be advantageous, allowing enhanced replication or broader tropism of the reassortant virus (Garten et al., 2009) (Fig. 1-1A). The morphology of the factories is attributed to the association of the µ2 protein from each strain with microtubules ( Parker et al., 2002 ).
Packaging a segmented viral genome is a complex process
- Packaging of segmented viral genomes often occurs through a core-filling or
- Packaging for viruses of the Reoviridae family requires RNA-RNA
- Packaging for viruses of the Reoviridae family requires RNA-protein
- Reoviridae family viruses are hypothesized to use an “all-or-nothing”
The packaging of multi-segment viral genomes is often done via a 'core-filling' model and a 'coordinated' model (McDonald et al., 2016). Rotavirus genome segments similarly rely on trans-segment interactions to form a packageable RNA complex (Fajardo et al., 2017).
Assembly requires the coordination of multiple processes, including transcription,
- Viruses with segmented genomes use various strategies to replicate within
- Reovirus undergoes transcription, translation, packaging, and assembly
- Reoviridae family VFs are established by viral nonstructural proteins and
- Reoviridae family virus factories are motile and display liquid-like properties
- Host responses to infection can determine success of viral infection
- Virus-virus interactions can influence coinfection potential
Specifically, mutations in the RNA-binding domain of σNS prevent access of +RNA to the VF (Lee et al., 2021). In the absence of segment mismatches, co-infected influenza A viruses readily rearrange genome segments (Marshall et al., 2013).
Summary
Reovirus and other members of the Reoviridae family compartmentalize replication within cytoplasmic VFs ( Tenorio et al., 2019 ). However, the frequency of reassortment is dramatically reduced with greater delay to superinfection (Marshall et al., 2013).
REOVIRUS LOW-DENSITY PARTICLES PACKAGE CELLULAR RNA
Introduction
Newly assembled core particles undergo secondary transcription, synthesizing additional viral RNA within virus factories ( Miller et al., 2010 ). Rotavirus nonstructural protein NSP2 binds viral +RNA, affects its structure, and is predicted to aid virus assembly (Borodavka, Desselberger, & Patton, 2018; Borodavka et al., 2017).
Coauthor Contributions
Host RNA selection by TC particles was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains independently of the viral RdRp. Although the precise features of host RNA that facilitate packaging into TC particles remain to be elucidated, these findings.
Results
- Reovirus top component particles are less infectious than virions
- Reovirus particles contain viral double-stranded RNA
- Top component particles contain host RNA
- The viral polymerase fails to confer complete host RNA packaging specificity
Percentage of viral and cellular reads from rsT1L-infected L cells and rsT1L or rsT3DIT1L1-TC particles or virions. Of the 34 host genes significantly enhanced in rsT1L TC particles relative to mock TC particles, none were. Whereas almost all viral reads in rsT1L virions and TC particles mapped to segments in the expected percentages based on length, .
Discussion
It is not clear why TC particles do not package the full set of viral genome segments. The packaging of viral and host RNA by rsT1L and rsT3DIT1L1 virions and TC particles showed marked differences. In addition to highly structured, non-polyadenylated cellular RNAs, polyadenylated host transcripts were packaged into TC particles, particularly rsT3DIT1L1 TC particles (Table 2-3).
Summary
Recent work suggests that VF morphology is not an important determinant of reassortment frequency during concurrent co-infection (Hockman et al., 2022). In addition, VFs act as sites of reovirus genome packaging and new particle assembly (Miller et al., 2010). Reassortment frequency may vary depending on the host species (Postnikova et al., 2021; Lin et al., 2017).
REOVIRUS EFFICIENTLY REASSORTS GENOME SEGMENTS DURING
Introduction
In particular, it is well known that rearrangement events can disrupt critical interactions between viral RNA and proteins, leading to virus progeny that are less fit than parent viruses or completely non-viable ((White, Steel, and Lowen, 2017; Huang et al. , 2008; Lubeck, Palese and Schulman, 1979; Nibert, Margraf and Coombs, 1996); discussed in White and Lowen, 2018)). Reovirus replication occurs in cytoplasmic virus factories (VFs), which are composed of interactions between nonstructural proteins µNS and σNS and function as the primary site of viral positive RNA (+RNA) synthesis, genome packaging, and new particle assembly. Becker, Peters and Dermody, 2003; Miller et al., 2010). VFs first appear as small, pointed bodies in the cytoplasm, but fuse and enlarge as replication progresses ( Ooms et al., 2010 ; Bussiere et al., 2017 ).
Coauthor Contributions
Finally, the exclusion of superinfection is unlikely to affect reassortment during a single replication cycle, but may affect reassortment potential in certain contexts.
Results
- A genetically barcoded reovirus displays identical replication kinetics and can
- Reovirus reassortment occurs frequently during simultaneous coinfection
- RNA abundance fails to explain reassortment frequency during coinfection. 76
- Viral RNA abundance correlates with reassortment frequency during
- Branched DNA FISH enables specific detection of WT and BC +RNA
- VFs are unlikely to influence reovirus reassortment frequency during
- T3D I reovirus primary infection can limit superinfection
Red lines indicate BC genome segment. co-infected with WT and BC reovirus at an MOI of or 100 PFU per cell per virus before quantifying reassortment frequency using HRM. Abundance of superinfecting virus RNA decreases with longer time to superinfection and correlates with reassortment frequency. The proportion of +RNA from the primary infecting virus localized to VFs remained constant at each time point of superinfection (Figs. 3-6H).
Discussion
Since the beginning of our study, others have also observed that reovirus +RNA localizes to both VFs and the cytoplasm ( Lee et al., 2021 ). Finally, rotavirus viroplasmas lose their fluid-like properties later during infection, coinciding with the phosphorylation of NSP5 ( Geiger et al., 2021 ). Recent findings suggest that rotavirus packages both capped and uncapped RNA ( Moreno-Contreras et al., 2022 );
Summary
The magnitude of the host response to reovirus infection varies depending on the infecting strain. Thus, both type I and type III interferon responses are critical for protection of the host against reovirus infection. Together, these data suggest that amplification of the interferon response through IFNAR likely contributes to reovirus superinfection clearance in vitro.
ANTIVIRAL RESPONSES DIFFERENTIALLY MEDIATE SUPERINFECTION
Introduction
Similar studies of rotavirus superinfection exclusion reached the same conclusion (Ramig, 1990); however, more recent evidence has indicated that rotavirus does limit superinfection, just not completely (Maeda et al., 2022). Specifically, T1L reovirus suppresses the induction of interferon-stimulated genes (ISGs) interferon regulatory factor 7 (IRF7) and signal transducer and activator of transcription 1 (STAT1), whereas T3D does not (Zurney et al., 2009), and significantly less interferon-β is produced in response to T1L infection than T3D (Stuart, Holm, & Boehme, 2018). At mucosal sites of infection such as the lungs and gut, type III interferon responses are more critical for protection against reovirus infection (Peterson. et al., 2019; Baldridge et al., 2017), whereas type I interferon is important in limiting reovirus . spread and infection of distal organs (Phillips et al., 2020; Johannson et al., 2007).
Coauthor Contributions
In this chapter, I attempted to determine the mechanism of reovirus superinfection exclusion in vitro and in vivo and assessed the impact of superinfection exclusion on reassortment. I also determined the frequency of redistribution after co-infection and superinfection of mouse pups deficient in type I and type III interferon receptors. This may provide indirect evidence to rule out superinfection in vivo and suggests that ablation of type I and type III interferon responses in the intestine is not sufficient to restore co-infection and redistribution.
Results
- Type I interferon drives reovirus superinfection exclusion in vitro
- Type I and type III interferon signaling does suppress reovirus reassortment
- Reovirus reassortants were not detected during superinfection in vivo
To determine whether interferon signaling affects reovirus reassortment in vivo, I quantified the reassortment frequency from intestinal homogenates of 3-day-old wild-type, IFNAR KO, and interferon lambda receptor (IFNLR) KO B6 mice after a 48-h co-infection with WT and BC T3DI reoviruses. Thus, type I interferon is a driver of reovirus superinfection clearance in vitro, but type I and type III interferon signaling does not affect intestinal reassortment frequency during co-infection. Therefore, I sought to determine whether type I and type III interferon signaling could affect the reassortment rate during superinfection in vivo.
Discussion
Chapter 3 I present data indicating that during superinfection in vitro, the abundance of superinfecting virus decreases in accordance with reassortment frequency (Figs. 3-4B,C). In contrast, although the superinfecting virus is clearly present during superinfection in wild-type and IFNLR KO mice, no rearrangement was detected (Fig. 4-3A-C). Why the mechanism of superinfection exclusion in vivo might differ from that observed in vitro is an open question.
Summary
Efficient packaging of the viral genome is critical for the formation of infectious virus particles. 2004) Identification of the 5' sequences required for incorporation of an engineered ssRNA into the Reovirus genome. 1968) Regulation of transcription of the reovirus genome. 1977) Molecular basis of reovirus virulence: Role of the SI gene.
MATERIALS AND METHODS
Cell culture and antibodies
Baby hamster kidney cells expressing bacteriophage T7 RNA polymerase under the control of a cytomegalovirus promoter (BHK-T7; Komoto et al. 2014) were maintained in Dulbecco's minimum essential medium (DMEM; Corning) supplemented with 5% FBS (Gibco) kept and were treated with 1 mg/ml Geneticin (Gibco) every other passage. All media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/mL streptomycin (Corning), and 25 ng/ml amphotericin B. Rabbit polyclonal reovirus antiserum and rabbit σNS-specific antiserum, (Becker. 2001) were gifts from Dr.
Viruses
BHK-T7 cells at ~50% confluency in 6-well plates were transfected with 0.8 µg of each plasmid encoding ten segments of the T1L genome, wild-type T3DI, or the barcoded genome. For viruses with swapped L1 and S1 segments between T1L and T3DI viruses, RNA was extracted from virus stocks, and the identities of L1 and S1 were verified by Sanger sequencing. The presence of the designed barcoded mutations in the rsT3DI BC virus was confirmed by Sanger sequencing after RNA extraction from viral stocks with TRIzol (Invitrogen) and.
Reovirus particle enrichment
Virus particle normalization
Bioanalyzer analysis
Library preparation and next-generation RNA-sequencing
Benzonase was inactivated with 0.5 M EDTA (pH 8.0) and RNA was extracted from virions and TC particles by TRIzol (Invitrogen) extraction per. Briefly, ribosomal RNA was removed from L cell samples via RNase H and DNase I digestion, and RNA was then purified using RNAClean XP beads (Beckman Coulter). RNA was fragmented before first-strand and second-strand synthesis and RNAClean XP purification.
Next-generation sequencing analysis
PCR enrichment of adapter-ligated DNA was performed using NEBNext Multiplex Oligos for Illumina (New England Biolabs) to produce Illumina-ready libraries. Illumina-ready libraries were sequenced by 150 base pair paired-end sequencing on the NovaSeq 6000 Sequencing System (Illumina). To illustrate Illumina reads mapped to the plus and minus strand of each viral genome segment, bam files were converted to bedGraph files using bedtools (scaled to one million with bedtools using command 'bedtools genomecov -bg - pc -scale Quinlan and Hall, 2010).
Quantitative reverse transcription PCR (RT-qPCR)
To quantify viral transcripts from coinfected and superinfected cells, L cell monolayers were disrupted by scraping and were pelleted at 200 x g for 5 min before RNA was isolated from 2 x 105 cells using the RNeasy Plus Mini RNA extraction kit (Qiagen) according to the manufacturer's instructions protocol. For viral RNA standards, RNA extracted from WT and BC T3DI viruses was purified by cesium chloride gradient ultracentrifugation, normalized based on concentration, serially diluted to obtain target standard curve concentrations, and reverse transcribed. To calculate the concentration of RNA, the concentration of total RNA from standard curve stocks was quantified by Nanodrop.
Fluorescent focus assay
Negative-stain electron microscopy
Multi-step replication
Coinfection experiments
Superinfection experiments
Viral +RNA was quantified by RT-qPCR, and the parent segment origin was determined by HRM analysis, as described below.
Mouse coinfection and superinfection experiments
Viral genotyping by high resolution melt analysis
Melting curves were generated after cDNA amplification and genotyping of WT and BC was performed with High Resolution Melting Software ver. Clones that consistently melted at a temperature between the expected melting temperatures for the wild-type and barcoded genotypes, giving an ambiguous parental genotype, were removed from the analysis. Thus, clones of an unclear parental origin may represent plaques that have formed from aggregated WT and BC virions.
Branched DNA FISH staining and image analysis of +RNA and VFs
VFs identified in this manner were visually confirmed to represent true staining before determining whether VFs contained WT and BC+RNA. To determine whether VFs contained +RNA from WT and BC, background was subtracted and images on the WT +RNA (AlexaFluor488) and BC +RNA (AlexaFluor647) channels were determined. The regions of interest identifying VFs from each cell were overlaid on the thresholded +RNA channels and the mean fluorescence intensity of the +RNA signal within VFs was quantified.
Statistical analysis
To determine whether primary infection limited the abundance of superinfecting virus transcripts at a 24 h superinfection time point, statistical significance was determined by unpaired t test. For experiments comparing transcript abundance in mock primary infection and primary BC infection over time, statistically. In Chapter 4, to determine whether abundance of superinfecting virus transcripts was altered after T1L or T3D primary infection relative to mock primary infection in wild-type or IFNAR KO SVECs, one-way ANOVA analysis with Tukey's multiple.
Data Availability
Given that many of the host RNAs packaged by the above component share structural features with reovirus + RNA transcripts, it is possible that the packaging of these host RNAs interferes with reovirus genomic packaging. For example, many viruses in the Reoviridae family replicate within protein VFs that are similar to those established by reovirus (Tenorio et al., 2019). 1974) Transcriptional control of the reovirus genome. 2009) Emergence and pandemic potential of H1N1 swine influenza virus. 2004).
Summary and Future Directions
Introduction
Recent in vitro packaging studies of bluetongue virus and rotavirus have significantly improved our understanding of the genome packaging requirements for these viruses; however, prior to my dissertation research, little was known about the packaging specificity and ability of these viruses to package non-viral RNA. At the beginning of my study, little was understood about the contribution of these virus-host and virus-virus interactions to reovirus redistribution. Reovirus packaging is precise and only allows the introduction of new types of RNA in the absence of a genome.
Reovirus packaging is precise and only allows for introduction of novel RNA
A major open question related to host RNA packaging by top component is what impact host RNA packaging has on viral genome packaging. To directly test the influence of host RNA packaging on the ability of reovirus to package its viral genome, host RNAs packaged into top component particles could be transiently overexpressed by transfection just prior to reovirus infection. Therefore, packaging of host RNA into peak component particles is unlikely to affect host cells that take up the peak.
Reassortment is an efficient process for many viruses with segmented genomes
Incompatibilities between parental viruses were a well-established constraint on recombination when I began my studies (Lubeck, Palese, and Schulman, 1979; Wenske et al., 1985); Quantification of influenza A virus reassortment in the absence of segment mismatches showed that reassortment occurs frequently during simultaneous coinfection and with a time delay to superinfection of up to 8 h (Marshall et al., 2013). These findings are supported by fluorescent in situ hybridization studies, which show that the majority of reovirus RNA is not localized to the VF (Lee et al., 2021).
Superinfection exclusion may pose a barrier to reovirus reassortment
Given that reovirus induces the expression of type 1 interferon, this seemed an obvious candidate for potentially ruling out superinfections. The observation that reassortants were not detected during superinfection in vivo is not necessarily indicative of superinfection exclusion. Branched DNA FISH probes specific for coinfecting viruses, which were developed as part of this thesis, combined with flow cytometry, would be useful tools in future studies of in vivo coinfection frequency and exclusion of superinfections.
Concluding Remarks
2013) Bunyaviridae viruses: all available isolates are reassortants. 2005) Carboxylic proximal regions of the reovirus nonstructural protein µNS necessary and sufficient for the formation of factory-like inclusions. 3' sequences required for incorporation of the constructed ssRNA into the reovirus genome. 2007) Characteristics of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that directs packaging and serotype restriction. 2007). Defective viral genomes are key factors in the virus-host interaction. 2017) Fitness costs of reallocation in human influenza.
