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

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Asha A. Philip ◽  
Jacob L. Perry ◽  
Heather E. Eaton ◽  
Maya Shmulevitz ◽  
Joseph M. Hyser ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
African Swine Fever Virus ◽  
Genetically Engineered ◽  
Genome Segment ◽  
Fusion Product ◽  
Wild Type ◽  
Infected Cells

ABSTRACT 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.

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

2019 ◽  
Author(s):  
Asha A. Philip ◽  
Jacob L. Perry ◽  
Heather E. Eaton ◽  
Maya Shmulevitz ◽  
Joseph M. Hyser ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Genetically Engineered ◽  
Genome Segment ◽  
Fusion Product ◽  
Infected Cells

AbstractRotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV 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 that encodes full-length NSP3 fused to UnaG, a 139-aa 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 passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimersin vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that 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 deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool 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.ImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (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 likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

scholarly journals A Genetically Engineered Rotavirus NSP2 Phosphorylation Mutant Impaired in Viroplasm Formation and Replication Shows an Early Interaction between vNSP2 and Cellular Lipid Droplets

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Jeanette M. Criglar ◽  
Sue E. Crawford ◽  
Boyang Zhao ◽  
Hunter G. Smith ◽  
Fabio Stossi ◽  
...  
Keyword(s):  
Virus Replication ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Genetically Engineered ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Nonstructural Protein 2 ◽  
Infected Cells

ABSTRACT Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication in viroplasms requires interactions between virus nonstructural proteins NSP2 and NSP5, which are associated with components of lipid droplets (LDs). We previously identified two forms of NSP2 in RV-infected cells, a cytoplasmically dispersed form (dNSP2) and a viroplasm-specific form (vNSP2), which interact with hypophosphorylated and hyperphosphorylated NSP5, respectively, indicating that a coordinated phosphorylation cascade controls viroplasm assembly. The cellular kinase CK1α phosphorylates NSP2 on serine 313, triggering the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. Using reverse genetics, we generated a rotavirus with a phosphomimetic NSP2 (S313D) mutation to directly evaluate the role of CK1α NSP2 phosphorylation in viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation and in virus replication and interferes with wild-type RV replication in coinfection. Taking advantage of the delay in viroplasm formation, the NSP2 phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (i) viroplasm assembly correlates with NSP5 hyperphosphorylation and (ii) vNSP2 S313D colocalizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly. IMPORTANCE Reverse genetics was used to generate a recombinant rotavirus with a single phosphomimetic mutation in nonstructural protein 2 (NSP2 S313D) that exhibits delayed viroplasm formation, delayed replication, and an interfering phenotype during coinfection with wild-type rotavirus, indicating the importance of this amino acid during virus replication. Exploiting the delay in viroplasm assembly, we found that viroplasm-associated NSP2 colocalizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation and that NSP5 hyperphosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for interaction with lipid droplets and may be the virus factor that induces lipid droplet biogenesis in rotavirus-infected cells. Lipid droplets are cellular organelles critical for the replication of many viral and bacterial pathogens, and thus, understanding the mechanism of NSP2-mediated viroplasm/lipid droplet initiation and interaction will lead to new insights into this important host-pathogen interaction.

scholarly journals Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains

2002 ◽  
Vol 76 (7) ◽  
pp. 3461-3470 ◽  
Author(s):  
Catherine Eichwald ◽  
Fulvia Vascotto ◽  
Elsa Fabbretti ◽  
Oscar R. Burrone
Keyword(s):  
Virus Infection ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphorylation Sites ◽  
Kinase Activation ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione S-transferase-NSP5 mutants were produced in Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser153, Ser155, Ser163, and Ser165) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.

scholarly journals Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Morisada Hayakawa ◽  
Asuka Sakata ◽  
Hiroko Hayakawa ◽  
Hikari Matsumoto ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Fluorescent Protein ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Coagulation Factor Viii ◽  
Liver Sinusoidal Endothelial Cells ◽  
Genetically Engineered Mice

AbstractCoagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.

1991 ◽  
Vol 174 (6) ◽  
pp. 1629-1637 ◽  
Author(s):  
J H Cox ◽  
J R Bennink ◽  
J W Yewdell
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Antigen Presentation ◽  
Viral Proteins ◽  
Genetically Engineered ◽  
Class I ◽  
Wild Type ◽  
Histocompatibility Complex ◽  
Infected Cells ◽  
Lumenal Domain

The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.

scholarly journals Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats

2020 ◽  
Vol 101 (10) ◽  
pp. 1056-1068
Author(s):  
Linda J. Rennick ◽  
Sham Nambulli ◽  
Ken Lemon ◽  
Grace Y. Olinger ◽  
Nicholas A. Crossland ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Human Respiratory Syncytial Virus ◽  
Subtype B ◽  
Cotton Rats ◽  
Syncytial Virus ◽  
Subtype A

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

scholarly journals Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

2007 ◽  
Vol 81 (17) ◽  
pp. 9490-9501 ◽  
Author(s):  
Christine D. Krempl ◽  
Anna Wnekowicz ◽  
Elaine W. Lamirande ◽  
Giw Nayebagha ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Virulent Strain ◽  
Consensus Sequence ◽  
Cytoplasmic Tail ◽  
Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

scholarly journals Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

2015 ◽  
Vol 89 (9) ◽  
pp. 4907-4917 ◽  
Author(s):  
Anna M. Mielech ◽  
Xufang Deng ◽  
Yafang Chen ◽  
Eveline Kindler ◽  
Dorthea L. Wheeler ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protease Activity ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Viral Pathogenesis ◽  
Wild Type ◽  
Viral Protease ◽  
Novel Approach ◽  
Protease Stability

ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document