Collection of Recombinant Rotaviruses Expressing Fluorescent Reporter Proteins

Philip, Asha A.; Herrin, Brittany E.; Garcia, Maximiliano L.; Abad, Andrew T.; Katen, Sarah P.; Patton, John T.

doi:10.1128/mra.00523-19

Cited by 20 publications

(24 citation statements)

References 13 publications

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“…1). The S sequences were inserted into the pT7/NSP3SA11 vector at the same site as used before in the production of recombinant SA11 (rSA11) rotaviruses expressing FPs (15)(16)(17).…”

Section: Modified Segment 7 (Nsp3) Expression Vectors Containing Sarsmentioning

confidence: 99%

“…Advances in rotavirus reverse genetics technologies have allowed the generation of recombinant rotaviruses that serve as expression platforms of heterologous proteins (12)(13)(14)(15)(16)(17)(18)(19). The rotavirus genome consists of 11 segments of dsRNA, with a total size of ~18.6 kbp for group A strains (rotavirus species A) typically associated with pediatric AGE (20).…”

Section: Introductionmentioning

confidence: 99%

“…Through this approach, well-growing genetically-stable recombinant rotaviruses have been generated that express NSP3 and one or more fluorescent proteins (FPs) [e.g., mRuby (red), UnaG (green), TagBFP (blue), etc.] from segment 7, an advance allowing study of rotavirus biology by live cell imaging (15). The NSP3 product of these recombinant viruses is functional, capable of dimerization and inducing the nuclear accumulation of the cellular poly(A)-binding protein (16,17).…”

Section: Introductionmentioning

confidence: 99%

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Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Preprint

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Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment.

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“…1). The S sequences were inserted into the pT7/NSP3SA11 vector at the same site as used before in the production of recombinant SA11 (rSA11) rotaviruses expressing FPs (15)(16)(17).…”

Section: Modified Segment 7 (Nsp3) Expression Vectors Containing Sarsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Preprint

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“…We infected MA104-GCaMP5G cells with SA114F or with a recombinant SA11 cl. 3 expressing mRuby from the NSP3 gene (SA11cl3-mRuby) 28 . Cells were infected at MOI of 10, 1, or 0.1, and we performed time-lapse Ca 2+ imaging and single-cell analysis of the resulting Ca 2+ signaling.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, the NSP4 open reading frame (ORF) in the pt7/NSP3 plasmid was replated with an ORF encoding NSP3 fused downstream to FLAG-tagged mRuby3. To promote the translation of NSP3 and mRuby3 as separate proteins, we inserted a teschovirus 2A-like stop-restart translation element between the NSP3 and FLAG-tagged mRuby3 coding sequences 28 . The SA11-mRuby virus was generated by co-transfection of BHK-T7 cells with pT7 plasmids expressing RV plus-sense RNAs along with a plasmid expressing the African swine fever virus NP868R capping enzyme from a CMV promoter.…”

Section: Methodsmentioning

confidence: 99%

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Chang‐Graham

Perry

Strtak

et al. 2019

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28 Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating 29 cytosolic calcium ([Ca 2+ ]cyt) and decreasing endoplasmic reticulum (ER) stores. While 30 an overall, monophasic increase in [Ca 2+ ]cyt during RV infection has been shown, the 31 nature of the RV-induced aberrant calcium signals and how they manifest over time at 32 the single-cell level have not been characterized. Thus, we generated cell lines and 33 human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted 34 genetically-encoded calcium indicators to characterize calcium signaling throughout RV 35 infection by time-lapse imaging. We found that RV induces highly dynamic [Ca 2+ ]cyt 36 signaling that manifest as hundreds of discrete [Ca 2+ ]cyt spikes, which increase during 37 peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca 2+ ]cyt 38 spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced 39 [Ca 2+ ]cyt spikes were primarily from ER calcium release and were attenuated by 40 inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs 41 also exhibited prominent [Ca 2+ ]cyt spikes that were attenuated by inhibiting SOCE, 42 underlining the relevance of these [Ca 2+ ]cyt spikes to gastrointestinal physiology and 43 role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca 2+ ]cyt 44 by dynamic Ca 2+ signaling, establishes a new, paradigm-shifting understanding of the 45 spatial and temporal complexity of virus-induced Ca 2+ signaling. 46 47 48 Eukaryotic signal transduction pathways employ a variety of signaling molecules 49 to regulate cellular processes. Calcium (Ca 2+ ) is one of the most ubiquitous secondary 50 messengers in the cell, which tightly regulates Ca 2+ movement through the coordinated 51 function of Ca 2+ channels, transporters, and pumps. Since Ca 2+ signaling modulates a 52 wide array of cellular processes, it is not surprising that many different viruses exploit 53 Ca 2+ signaling to facilitate their replication, and the resulting dysregulation of Ca 2+ 54 signaling causes pathogenesis. Rotavirus (RV), a member of the Reoviridae family, is 55 one of the first viruses shown to elevate cellular Ca 2+ levels and has become a widely-56 used model system to characterize mechanisms by which viruses dysregulate host Ca 2+ 57 homeostasis 1 . RV is a clinically important enteric virus that causes severe diarrhea and 58 vomiting in children, resulting in over approximately 258 million diarrhea episodes and 59 198,000 deaths in 2016 2 . Hyperactivation of cyclic nucleotide (e.g., cAMP/cGMP) and 60 Ca 2+ signaling pathways is a common strategy among enteric pathogens 3 . Thus, 61 understanding how RV exploits Ca 2+ signaling is key to understanding and combating 62 RV-induced diarrhea. 63 RV was first reported to elevate cytosolic [Ca 2+ ] by Michelangeli et al. (1991), 64 which stimulated subsequent research into how RV alters cellular Ca 2+ levels 4 . RV 65 causes a 2-fold steady-state increase in ...

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