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.

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 Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

scholarly journals The Expression Level of the 3a Movement Protein Determines Differences in Severity of Symptoms Between Two Strains of Tomato Aspermy Cucumovirus

1997 ◽  
Vol 10 (2) ◽  
pp. 171-179 ◽  
Author(s):  
Ignacio M. Moreno ◽  
Juan José Bernal ◽  
Blanca García de Blas ◽  
Emilio Rodriguez-Cerezo ◽  
Fernando García-Arenal
Keyword(s):  
Movement Protein ◽  
Stop Codon ◽  
Site Directed Mutagenesis ◽  
In Vitro Translation ◽  
Protein Accumulation ◽  
Cdna Clones ◽  
Post Inoculation ◽  
Mild Phenotype ◽  
Systemic Leaf ◽  
Chlorotic Mottle

Two strains of tomato aspermy cucumovirus, 1-TAV and V-TAV, differ in the severity of the symptoms induced in Nicotiana tabacum: 1-TAV induces a severe chlorotic mottle that appears 5 days post inoculation (d.p.i.) in the second systemic leaf, while V-TAV-infected plants show a mild chlorotic mottle, unevenly distributed in the leaf lamina, that appears 7 d.p.i. in the third or fourth systemic leaf. The manipulation of full-length cDNA clones giving infectious transcripts of V-TAV RNAs 1, 2, and 3 and 1-TAV RNA 3 revealed that the slow, mild phenotype of V-TAV maps to the movement protein (MP) gene. By site-directed mutagenesis it was further shown that this phenotype co-segregates with a single nucleotide substitution that introduces an in-frame UAA stop codon at the fourth position of the MP open reading frame of V-TAV. The presence of this stop codon results in a diminished expression of the MP in both tobacco protoplasts and leaves. Analyses of the progress of infection and of the time course of MP and coat protein accumulation show that the low level of MP in V-TAV-infected leaves limits the rate of cell-to-cell movement and leads to the mild phenotype. Data from the infectivity of RNA 3 transcripts with or without this stop codon, plus data from in vitro translation of virion or transcript RNA 3, suggest that the small amount of MP observed in V-TAV-infected leaves is expressed from a minor RNA 3 subpopulation lacking the stop codon.

scholarly journals Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2142-2150 ◽  
Author(s):  
R A Levine ◽  
G J LaRosa ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Cdna Library ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Cdna Clones ◽  
Dibutyryl Cyclic Amp

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA

1990 ◽  
Vol 10 (5) ◽  
pp. 2315-2326
Author(s):  
J H Teruya ◽  
S Y Kutsunai ◽  
D H Spear ◽  
P A Edwards ◽  
C F Clarke
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Farnesyl Pyrophosphate ◽  
S1 Nuclease ◽  
Testis Cdna ◽  
Low Efficiency

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

Characterization of the rabbit renal Na(+)-dicarboxylate cotransporter using antifusion protein antibodies

1996 ◽  
Vol 271 (6) ◽  
pp. C1808-C1816 ◽  
Author(s):  
A. M. Pajor ◽  
N. Sun
Keyword(s):  
Fusion Protein ◽  
Brush Border ◽  
Xenopus Oocytes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Site Directed Mutagenesis ◽  
In Vitro Translation ◽  
Western Blots ◽  
Renal Brush Border Membrane

Polyclonal antibodies were prepared against the rabbit renal Na(+)-dicarboxylate cotransporter, NaDC-1. The antibodies were raised in chickens against a fusion protein consisting of a 60-amino acid peptide from NaDC-1 and glutathione S-transferase. These antibodies specifically recognized the fusion protein in Western blots and could immunoprecipitate the full-length NaDC-1 after in vitro translation. The antifusion protein antibodies specifically recognized a protein of 63 kDa in rabbit renal brush-border membrane vesicles (BBMV), similar to the predicted mass of 66 kDa. Two proteins of 57 and 115 kDa were recognized in rabbit intestinal brush-border membranes. A protein of 66 kDa was recognized in Xenopus oocytes injected with NaDC-1 cRNA. Enzymatic deglycosylation of rabbit renal BBMV resulted in a decrease in mass by 11 kDa, consistent with N-glycosylation at a single site. Site-directed mutagenesis of the two consensus sequences for N-glycosylation in the NaDC-1 cDNA showed that Asn-576, located near the COOH-terminal, is glycosylated. The nonglycosylated mutant of NaDC-1 exhibited 50% of wild-type succinate transport activity when expressed in Xenopus oocytes, suggesting that glycosylation is not essential for function. The revised secondary structure model of NaDC-1 contains 11 putative transmembrane domains and an extracellular glycosylated COOH-terminal.

Expression and processing of the canine calicivirus capsid precursor

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 195-199 ◽  
Author(s):  
Yuichi Matsuura ◽  
Yukinobu Tohya ◽  
Mihoko Onuma ◽  
Frank Roerink ◽  
Masami Mochizuki ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Mammalian Cells ◽  
Expression System ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Feline Calicivirus ◽  
Putative Cleavage Site ◽  
Mammalian Expression ◽  
Capsid Precursor ◽  
Infected Cells

The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase.

Sign in / Sign up

Export Citation Format

Share Document