scholarly journals Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro

2001 ◽  
Vol 82 (7) ◽  
pp. 1785-1790 ◽  
Author(s):  
Karma Carrier ◽  
Yu Xiang ◽  
Hélène Sanfaçon
Keyword(s):  
Cleavage Site ◽  
Terminal Region ◽  
Ringspot Virus ◽  
Protein Domain ◽  
Site Directed Mutagenesis ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
Tomato Ringspot Virus ◽  
Definition Of

The proteinase of Tomato ringspot virus (genus Nepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using an in vitro processing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q301/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.

scholarly journals Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro

2000 ◽  
Vol 81 (11) ◽  
pp. 2771-2781 ◽  
Author(s):  
Aiming Wang ◽  
Hélène Sanfaçon
Keyword(s):  
Cleavage Site ◽  
Terminal Region ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
N Terminus ◽  
Partial Cdna ◽  
Coding Capacity

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q423/G and Q620/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.

scholarly journals Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

1998 ◽  
Vol 72 (4) ◽  
pp. 3051-3059 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Svetlana A. Sosnovtseva ◽  
Kim Y. Green
Keyword(s):  
Cleavage Site ◽  
Site Directed Mutagenesis ◽  
In Vitro Translation ◽  
Feline Calicivirus ◽  
Cdna Clones ◽  
Bacterial Cells ◽  
Structural Protein ◽  
Critical Determinant ◽  
Capsid Precursor

ABSTRACT Feline calicivirus (FCV), a member of theCaliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates fortrans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

scholarly journals Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

2006 ◽  
Vol 80 (16) ◽  
pp. 7816-7831 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Gaël Belliot ◽  
Kyeong-OK Chang ◽  
Victor G. Prikhodko ◽  
Larissa B. Thackray ◽  
...  
Keyword(s):  
Cell Culture ◽  
Caspase 3 ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Cleavage Sites ◽  
Murine Norovirus ◽  
Permissive Cell ◽  
Additional Processing ◽  
Infected Cells

ABSTRACT Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

scholarly journals The p23 Protein of Hibiscus Chlorotic Ringspot Virus Is Indispensable for Host-Specific Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12312-12319 ◽  
Author(s):  
Xiao-Zhen Liang ◽  
Andrew P. Lucy ◽  
Shou-Wei Ding ◽  
Sek-Man Wong
Keyword(s):  
Viral Replication ◽  
Fluorescent Protein ◽  
Chenopodium Quinoa ◽  
Hibiscus Cannabinus ◽  
Ringspot Virus ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Reading Frame

ABSTRACT Hibiscus chlorotic ringspot virus (HCRSV) possesses a novel open reading frame (ORF) which encodes a putative 23-kDa protein (p23). We report here the in vivo detection of p23 and demonstrate its essential role in viral replication. The expression of p23 could be detected in protein extracts from transfected kenaf (Hibiscus cannabinus L.) protoplasts and in HCRSV-infected leaves. Further, direct immunoblotting of infected kenaf leaves also showed the presence of p23, and transient expression in onion and kenaf cells demonstrated that the protein is distributed throughout the cell. Site-directed mutagenesis showed that mutations introduced into the ORF of p23 abolished viral replication in kenaf protoplasts and plants but not in Chenopodium quinoa L. The loss of function of the p23 mutant M23/S33-1 could be complemented in trans upon the induced expression of p23 from an infiltrated construct bearing the ORF (pCam23). Altogether, these results demonstrate that p23 is a bona fide HCRSV protein that is expressed in vivo and suggest that p23 is indispensable for the host-specific replication of HCRSV. In addition, we show that p23 does not bind nucleic acids in vitro and does not act as a suppressor of posttranscriptional gene silencing in transgenic tobacco carrying a green fluorescent protein.

scholarly journals A strain-specific segment of the RNA-dependent RNA polymerase of grapevine fanleaf virus determines symptoms in Nicotiana species

2013 ◽  
Vol 94 (12) ◽  
pp. 2803-2813 ◽  
Author(s):  
Emmanuelle Vigne ◽  
John Gottula ◽  
Corinne Schmitt-Keichinger ◽  
Véronique Komar ◽  
Léa Ackerer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Systemic Infection ◽  
Cdna Clones ◽  
Grapevine Fanleaf Virus ◽  
Rna Dependent Rna Polymerase ◽  
Coding Regions ◽  
Tomato Ringspot Virus ◽  
Quantitative Analyses

Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1EPol), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1EPol was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1EPol failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.

scholarly journals Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana

2002 ◽  
Vol 83 (8) ◽  
pp. 2085-2089 ◽  
Author(s):  
Simon Léonard ◽  
Joan Chisholm ◽  
Jean-François Laliberté ◽  
Hélène Sanfaçon
Keyword(s):  
Virus Genome ◽  
Translation Initiation Factor ◽  
Ringspot Virus ◽  
Initiation Factor ◽  
Virus Infectivity ◽  
Eukaryotic Translation ◽  
Tomato Ringspot Virus ◽  
Cellular Mrnas ◽  
Eukaryotic Translation Initiation

Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyvirus VPg and this interaction has been correlated with virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepovirus (Tomato ringspot virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m7GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the virus genome are discussed.

scholarly journals The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

2016 ◽  
Vol 473 (11) ◽  
pp. 1523-1536 ◽  
Author(s):  
Guillem Prats-Ejarque ◽  
Javier Arranz-Trullén ◽  
Jose A. Blanco ◽  
David Pulido ◽  
M. Victòria Nogués ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Kinetic Analysis ◽  
Cleavage Site ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cleavage Sites ◽  
Human Rnase ◽  
Dynamics Simulations

We describe the first human RNase 6 crystal structure in complex with sulfate anions. Kinetic analysis, site-directed mutagenesis and molecular dynamics simulations identified novel substrate recognition and cleavage sites.

Two distinct sites are essential for virulent infection and support of variant satellite RNA replication in spontaneous beet black scorch virus variants

2012 ◽  
Vol 93 (12) ◽  
pp. 2718-2728 ◽  
Author(s):  
Jin Xu ◽  
Xianbing Wang ◽  
Lindan Shi ◽  
Yuan Zhou ◽  
Dawei Li ◽  
...  
Keyword(s):  
Rna Virus ◽  
Point Mutations ◽  
Virus Evolution ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Cdna Clones ◽  
Sequence Alignments ◽  
Chenopodium Amaranticolor ◽  
Beet Black Scorch Virus

Spontaneous point mutations of virus genomes are important in RNA virus evolution and often result in modifications of their biological properties. Spontaneous variants of beet black scorch virus (BBSV) and its satellite (sat) RNA were generated from cDNA clones by serial propagation in Chenopodium amaranticolor and Nicotiana benthamiana. Inoculation with recombinant RNAs synthesized in vitro revealed BBSV variants with divergent infectious phenotypes that affected either symptom expression or replication of satRNA variants. Sequence alignments showed a correlation between the phenotypes and distinct BBSV genomic loci in the 3′UTR or in the domain encoding the viral replicase. Comparative analysis between a virulent variant, BBSV-m294, and the wild-type (wt) BBSV by site-directed mutagenesis indicated that a single-nucleotide substitution of a uridine to a guanine at nt 3477 in the 3′UTR was responsible for significant increases in viral pathogenicity. Gain-of-function analyses demonstrated that the ability of the BBSV variants to support replication of variant satRNAs was mainly determined by aa 516 in the P82 replicase. In this case, an arginine substitution for a glutamine residue was essential for high levels of replication, and alterations of other residues surrounding position 516 in the wtBBSV isolate led to only minor phenotypic effects. These results provide evidence that divergence of virus functions affecting pathogenicity and supporting parasitic replication can be determined by a single genetic site, either a nucleotide or an amino acid. The results suggest that complex interactions occur between virus and associated satRNAs during virus evolution.

scholarly journals A Novel Rice Curl Dwarf-Associated Picornavirus Encodes a 3C Serine Protease Recognizing Uncommon EPT/S Cleavage Sites

2021 ◽  
Vol 12 ◽  
Author(s):  
Tianze Zhang ◽  
Chenyang Li ◽  
Mengji Cao ◽  
Dan Wang ◽  
Qi Wang ◽  
...  
Keyword(s):  
Serine Protease ◽  
Genome Organization ◽  
Cleavage Site ◽  
Cysteine Proteases ◽  
Cleavage Sites ◽  
Rna Seq ◽  
3C Protease ◽  
Wide Range ◽  
Sequence Identities

Picornaviruses cause diseases in a wide range of vertebrates, invertebrates and plants. Here, a novel picornavirus was identified by RNA-seq technology from rice plants showing dwarfing and curling symptoms, and the name rice curl dwarf-associated virus (RCDaV) is tentatively proposed. The RCDaV genome consists of an 8,987 nt positive-stranded RNA molecule, excluding a poly(A) tail, that encodes two large polyproteins. Using in vitro cleavage assays, we have identified that the RCDaV 3C protease (3Cpro) as a serine protease recognizes the conserved EPT/S cleavage site which differs from the classic Q(E)/G(S) sites cleaved by most picornaviral 3C chymotrypsin-like cysteine proteases. Therefore, we comprehensively deciphered the RCDaV genome organization and showed that the two polyproteins of RCDaV can be cleaved into 12 mature proteins. We found that seven unclassified picornaviruses also encode a 3Cpro similar to RCDaV, and use the highly conserved EPT/S as the cleavage site. The precise genome organizations of these viruses were illustrated. Moreover, RCDaV and the seven unclassified picornaviruses share high sequence identities and similar genome organizations, and cluster into a distinct clade in the order Picornavirales. Our study provides valuable information for the understanding of picornaviral 3Cpros, deciphers the genome organization of a few relatively obscure picornaviruses, and lays the foundation for further pathogenesis research on these viruses.

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