scholarly journals Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells

2005 ◽  
Vol 79 (9) ◽  
pp. 5363-5373 ◽  
Author(s):  
Julie R. Harris ◽  
Vincent R. Racaniello
Keyword(s):  
Amino Acid ◽  
Viral Replication ◽  
Host Cell ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Viral Receptor ◽  
Viral Growth ◽  
L Cells ◽  
Cell Components ◽  
Mouse Cells

ABSTRACT Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.

scholarly journals The Leader Polypeptide of Theiler's Murine Encephalomyelitis Virus Is Required for the Assembly of Virions in Mouse L Cells

2000 ◽  
Vol 74 (2) ◽  
pp. 875-882 ◽  
Author(s):  
Cyrus Badshah ◽  
Miriam A. Calenoff ◽  
Kathleen Rundell
Keyword(s):  
Protein Synthesis ◽  
Virus Assembly ◽  
Viral Proteins ◽  
Viral Rna ◽  
Theiler's Murine Encephalomyelitis Virus ◽  
Viral Protein Synthesis ◽  
Theiler’S Murine Encephalomyelitis Virus ◽  
L Cells ◽  
Encephalomyelitis Virus ◽  
Mouse Cells

ABSTRACT Deletion of the entire leader polypeptide of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) results in the production of an attenuated virus that grows in baby hamster kidney (BHK) cells but cannot grow at all in mouse L-929 cells. This study examined the reasons for the failure of dl-L, the GDVII variant that lacks the leader polypeptide, to grow in mouse cells. At low multiplicities of infection, it was difficult to detect any viral proteins in mouse cells. However, levels of positive- and negative-strand RNA molecules were only moderately reduced in these infections. Viral RNA showed no major defect in translatability, as the mutant viral RNA was nearly as efficient as that of the wild-type (WT) virus in directing protein synthesis in vitro in assays using extracts prepared from mouse L cells. Viral protein synthesis was detected indl-L-infected mouse cells as multiplicities of infection were increased and approached the levels observed in WT infections. Despite this, there was a total lack of virus production in high-multiplicity infections, and this was found to correlate with the failure of viral proteins and early virion precursors to assemble into virions in mouse cells. Thus, the inability of dl-L to grow in mouse cells reflects complex effects on various stages of the virus infection but is primarily a defect in virus assembly.

scholarly journals Changes in Rhinovirus Protein 2C Allow Efficient Replication in Mouse Cells

2003 ◽  
Vol 77 (8) ◽  
pp. 4773-4780 ◽  
Author(s):  
Julie R. Harris ◽  
Vincent R. Racaniello
Keyword(s):  
Cytopathic Effect ◽  
Cellular Proteins ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Viral Receptor ◽  
L Cells ◽  
Variant Virus ◽  
Efficient Replication ◽  
Mouse Cells

ABSTRACT Rhinovirus type 16 was found to replicate in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However, infection of these cells at a low multiplicity of infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1 L cells. Infection of mouse cells with 16/L leads to higher virus titers, increased production of RNA, and total cytopathic effect. Three amino acid changes were identified in the P2 region of virus 16/L, and the adaptation phenotype mapped to two changes in protein 2C. The characterization of a rhinovirus host range mutant will facilitate the investigation of cellular proteins required for efficient viral growth and the development of a murine model for rhinovirus infection.

SARS Virus Papain-Like Protease: A Mysterious Weapon

2020 ◽  
Author(s):  
Reza Nejat ◽  
Ahmad Shahir Sadr
Keyword(s):  
Innate Immunity ◽  
Pulmonary Fibrosis ◽  
Viral Replication ◽  
Host Cell ◽  
Adaptive Immunity ◽  
Virus Genome ◽  
Type I ◽  
Nonstructural Proteins ◽  
Irf3 Activation

Introduction: Papain-like protease (PLpro) of SARS-CoV in association with 3Chemotrypsin-like protease (3CLpro or Mpro) are two proteases which auto-proteolyze replicase polyproteins pp1a/pp1ab. These polyproteins are translated from ORF1a/ORF1b of the virus genome. Cleavage of pp1a/pp1ab releases nonstructural proteins of the virus which orchestrate viral replication. In addition, PLpro as a deubiquitinase and deISGylase modifies the proteins involved in recognition of the virus by the sensors of host cell innate immunity system. In this manner, the virus reforms the ubiquitination and ISGylation of the cell proteins to progress its own replication without any interference from host cell restrictive strategies against the viruses. Furthermore, PLpro blocks IRF3 activation independent of deubiquinating processes. Besides, PLpro induces pulmonary fibrosis through pathways involving ROS and MAPK. Conclusion: Inhibition of PLpro allows innate immunity to sense and react against the invasion of SARSCoV and to activate IRF3 to induce type I IFN expression. Thenceforth, proper development and signaling of innate immunity result in a long-term efficient cell/humoral adaptive immunity. Moreover, suppression of PLpro prevents cleavage of nsp3 and hence replication of the virus and through abolishing ubiquitinproteasome/MAPK/ERK- and ROS/MAPK-mediated pathways prevent pulmonary fibrosis.

scholarly journals Localization of Mouse Hepatitis Virus Nonstructural Proteins and RNA Synthesis Indicates a Role for Late Endosomes in Viral Replication

1999 ◽  
Vol 73 (9) ◽  
pp. 7641-7657 ◽  
Author(s):  
Yvonne van der Meer ◽  
Eric J. Snijder ◽  
Jessika C. Dobbe ◽  
Sibylle Schleich ◽  
Mark R. Denison ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Multilayered Structures ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Infected Cells

ABSTRACT The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5′-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.

scholarly journals In Silico studies of Natural compounds that inhibit SARS-CoV-2 Nucleocapsid Nsp1/Nsp3 proteins mediated Viral Replication and Pathogenesis

2020 ◽  
Author(s):  
Hemanth Kumar Manikyam
Keyword(s):  
Viral Replication ◽  
Host Cell ◽  
Cyclophilin A ◽  
Natural Compounds ◽  
Docking Studies ◽  
Boswellic Acid ◽  
Nonstructural Proteins ◽  
Nucleocapsid Proteins ◽  
Withanolide A ◽  
Cucurbitacin E

Abstract Highly Transmissible and pathogenic coronavirus that emerged in late December of 2019 caused Severe acute respiratory syndrome (SARS-CoV-2), which challenged human health and public safety. Severity of the disease depends on the viral load and the type of mutation that occurred in the coronavirus. Nonstructural proteins like, Nsp1, Nsp3, Nsp12 and Nsp13 including other viral proteins plays important role during viral replication life cycle. Viral Replication initiated by hacking the host cellular mechanism either by synergy or by suppression using nucleocapsid proteins of the virus. Spike (S) protein of the SARS-CoV-2 uses angiotensin-converting enzyme II (ACE2) and TRMPSS as a cell entry. Once virus enters host cell, nucleocapsid proteins along with its genome is releases from endosomes into cytosol of the host cell. Ca2+/CaM (Calmodulin)/Calcineurin complex of the host cell plays important role during viral replication which is mediated by nucleocapsid proteins of the virus. Nsp1/Nsp3 nonstructural proteins triggers synergetic activity with CD147/CyPA/HSPG pathway and TRMP2/ADPr/Ca+2 mediated Ca2+/CaM (Calmodulin)/Calcineurin synthesis and free radicle generation in mitochondria leading to viral replication and severe chemokine activation pathways. Docking studies were carried out to inhibit Cyclophilin A and TRMP2 proteins as drug targets. Natural compounds like Withanolide A, Columbin, Cucurbitacin E, Boswellic acid along with Cyclosporines, Vitamin E and N-Acetyl cysteine (NAC) were selected as ligands to study docking studies. Withanolide A and Cyclosporines had shown good inhibition activity against Cyclophilin A, whereas Columbin, Boswellic acid, Cucurbitacin E, Vitamin E and N-Acetyl cysteine (NAC) had shown inhibitory activity against TRMP2. Thus, we suggest conducting further studies to conclude above pathways mechanism and inhibitory effect of natural compounds against the Nsp1/Nsp3 mediated pathways Invitro and In vivo.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

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