A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

Mohanty, Sujit K.; Donnelly, Bryan; Dupree, Phylicia; Lobeck, Inna N.; Mowery, Sarah; Meller, Jarosław; McNeal, Monica; Tiao, Greg

doi:10.1128/jvi.00510-17

Cited by 14 publications

(12 citation statements)

References 56 publications

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“…African green monkey kidney cells, MA104 cells, (BioWhittaker, Walkersville, MD, USA) were cultured as described [ 16 ]. The simian rotavirus strain, RRV (generously provided by H. Greenberg, Stanford University, Palo Alto, CA, USA), was propagated in MA104 cells as previously described [ 11 , 17 ].…”

Section: Methodsmentioning

confidence: 99%

T-Bet Deficiency Attenuates Bile Duct Injury in Experimental Biliary Atresia

Mohanty

Donnelly

Temple

et al. 2021

Cells

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Biliary atresia (BA) is an obstructive neonatal cholangiopathy leading to liver cirrhosis and end stage liver disease. A Kasai portoenterostomy may restore biliary drainage, but most patients ultimately require liver transplantation for survival. At diagnosis, immune cells within the liver of patients with BA demonstrate a T-helper 1 (Th1) inflammatory profile similar to rhesus rotavirus (RRV)-infected mice livers developing BA. The transcription factor Tbx21 (T-bet) is essential for induction of a Th1 immune response in both the adaptive and innate immune system. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of BA. Infection of newborn T-bet knockout (KO) pups with RRV resulted in a decreased Th1 inflammatory chemokine/cytokine profile when compared to infected wild-type mice. Analysis of the mononuclear cells profile from T-bet KO mice revealed both a significant decrease in the total number of CD3, CD4, and CD8 T cells and their effector molecules granzyme A, perforin, and FasL. Even though the percentage of T-bet KO mice displaying symptoms of an obstructive cholangiopathy and overall mortality rate was not different compared to wild-type mice, the extrahepatic bile ducts of T-bet KO mice remained patent.

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Section: Methodsmentioning

confidence: 99%

T-Bet Deficiency Attenuates Bile Duct Injury in Experimental Biliary Atresia

Mohanty

Donnelly

Temple

et al. 2021

Cells

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“…This tripeptide "SRL" on RRV VP4 was found to bind specifically to the cholangiocyte membrane protein heat shock cognate 70 (Hsc70), defining a novel binding site governing VP4 attachment (57). To gain insight into the mechanisms involved upon VP4-mediated infection, a reverse genetics system was developed to create a mutant of RRV with a single amino acid change in the VP4 protein and compared to that of wild-type RRV (where the arginine "R" in "SRL" region was replaced with glycine "G") (58). The mutant virus, when injected to mice, demonstrated reduced symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia indicating the importance of "SRL" region (57).…”

Section: Evidence Of Viruses As a Causative Agent Of Bamentioning

confidence: 99%

Innate Immunity and Pathogenesis of Biliary Atresia

et al. 2020

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Biliary atresia (BA) is a devastating fibro-inflammatory disease characterized by the obstruction of extrahepatic and intrahepatic bile ducts in infants that can have fatal consequences, when not treated in a timely manner. It is the most common indication of pediatric liver transplantation worldwide and the development of new therapies, to alleviate the need of surgical intervention, has been hindered due to its complexity and lack of understanding of the disease pathogenesis. For that reason, significant efforts have been made toward the development of experimental models and strategies to understand the etiology and disease mechanisms and to identify novel therapeutic targets. The only characterized model of BA, using a Rhesus Rotavirus Type A infection of newborn BALB/c mice, has enabled the identification of key cellular and molecular targets involved in epithelial injury and duct obstruction. However, the establishment of an unleashed chronic inflammation followed by a progressive pathological wound healing process remains poorly understood. Like T cells, macrophages can adopt different functional programs [pro-inflammatory (M1) and resolutive (M2) macrophages] and influence the surrounding cytokine environment and the cell response to injury. In this review, we provide an overview of the immunopathogenesis of BA, discuss the implication of innate immunity in the disease pathogenesis and highlight their suitability as therapeutic targets.

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“…It remains unclear how much foreign sequence can be inserted into the segment 7 RNA, but our analysis thus far indicates that it is possible to generate well-replicating viruses carrying Ͼ500 bp of extra sequence. Given the remarkable flexibility in the ability of the rotavirus to accommodate changes in the size and sequence of its RNA noted thus far (6,9,12,(33)(34)(35), the virus may be a particularly empowering tool for elucidating the shared mechanisms used by the Reoviridae to package and to replicate their genomes. Plasmid construction.…”

Section: Figmentioning

confidence: 99%

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

Philip

Perry

Eaton

et al. 2019

J Virol

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Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo. Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA. IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

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A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

Cited by 14 publications

References 56 publications

T-Bet Deficiency Attenuates Bile Duct Injury in Experimental Biliary Atresia

T-Bet Deficiency Attenuates Bile Duct Injury in Experimental Biliary Atresia

Innate Immunity and Pathogenesis of Biliary Atresia

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

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