scholarly journals Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase

2010 ◽  
Vol 84 (14) ◽  
pp. 7325-7336 ◽  
Author(s):  
Shouguo Fang ◽  
Hongyuan Shen ◽  
Jibin Wang ◽  
Felicia P. L. Tay ◽  
Ding Xiang Liu
Keyword(s):  
Infectious Bronchitis Virus ◽  
Nonstructural Protein ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Genetic Studies ◽  
Subgenomic Rna ◽  
Infectious Bronchitis ◽  
Growth Defects ◽  
Cleavage Efficiency

ABSTRACT Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.

scholarly journals Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

2006 ◽  
Vol 80 (11) ◽  
pp. 5413-5422 ◽  
Author(s):  
Aleksei Lulla ◽  
Valeria Lulla ◽  
Kairit Tints ◽  
Tero Ahola ◽  
Andres Merits
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Cleavage Site ◽  
Semliki Forest Virus ◽  
Nonstructural Protein ◽  
Cleavage Sites ◽  
Vitro Assay ◽  
Virus Life Cycle ◽  
Cleavage Efficiency

ABSTRACT The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

scholarly journals Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1102
Author(s):  
Phoebe Stevenson-Leggett ◽  
Sarah Keep ◽  
Erica Bickerton
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Serial Passage ◽  
In Ovo ◽  
Infectious Bronchitis ◽  
Protease Treatment ◽  
Cellular Tropism ◽  
Passage Cell

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

scholarly journals Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Paula F. Zamora ◽  
Liya Hu ◽  
Jonathan J. Knowlton ◽  
Roni M. Lahr ◽  
Rodolfo A. Moreno ◽  
...  
Keyword(s):  
Virus Replication ◽  
Viral Genome ◽  
Nonstructural Protein ◽  
Dsrna Virus ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Content Type ◽  
The Family ◽  
Viral Genome Replication

ABSTRACTViral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, usingin vitroand cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCEFollowing infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the familyReoviridaeencode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the familyReoviridaediverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Usingin vitroand cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the familyReoviridaeassort and replicate their genomes.

scholarly journals N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity

2020 ◽  
Vol 101 (6) ◽  
pp. 599-608
Author(s):  
K. M. Bouwman ◽  
N. Habraeken ◽  
A. Laconi ◽  
A. J. Berends ◽  
L. Groenewoud ◽  
...  
Keyword(s):  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Glycosylation Site ◽  
Host Cells ◽  
Receptor Specificity ◽  
Knock Out ◽  
Recombinant Viruses ◽  
Infectious Bronchitis

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

scholarly journals Differential Sensitivity of Well-Differentiated Avian Respiratory Epithelial Cells to Infection by Different Strains of Infectious Bronchitis Virus

2010 ◽  
Vol 84 (17) ◽  
pp. 8949-8952 ◽  
Author(s):  
Sahar Abd El Rahman ◽  
Christine Winter ◽  
Ali El-Kenawy ◽  
Ulrich Neumann ◽  
Georg Herrler
Keyword(s):  
Respiratory Tract ◽  
Infectious Bronchitis Virus ◽  
Bronchial Epithelium ◽  
Influenza Viruses ◽  
Differential Sensitivity ◽  
Respiratory Pathogens ◽  
Infectious Bronchitis ◽  
Well Differentiated ◽  
Different Strains

ABSTRACT Infectious bronchitis virus (IBV) is an avian coronavirus affecting the respiratory tract of chickens. To analyze IBV infection of the lower respiratory tract, we applied a technique that uses precision-cut lung slices (PCLSs). This method allows infection of bronchial cells within their natural tissue composition under in vitro conditions. We demonstrate that IBV strains 4/91, Italy02, and QX infect ciliated and mucus-producing cells of the bronchial epithelium, whereas cells of the parabronchial tissue are resistant to infection. This is the first study, using PCLSs of chicken origin, to analyze virus infection. PCLSs should also be a valuable tool for investigation of other respiratory pathogens, such as avian influenza viruses.

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