scholarly journals Rubella Virus Nonstructural Protein Protease Domains Involved in trans- and cis-Cleavage Activities

2000 ◽  
Vol 74 (12) ◽  
pp. 5412-5423 ◽  
Author(s):  
Yuying Liang ◽  
Jiansheng Yao ◽  
Shirley Gillam
Keyword(s):  
Rubella Virus ◽  
Nonstructural Protein ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Translation System ◽  
Nonstructural Proteins ◽  
Cleavage Activity ◽  
Domain Of Unknown Function

ABSTRACT Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5′-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3′-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH2-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and incis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- andtrans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- andtrans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not forcis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.

scholarly journals Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

scholarly journals Analysis of the 3′ cis-Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

2004 ◽  
Vol 78 (5) ◽  
pp. 2553-2561 ◽  
Author(s):  
Min-Hsin Chen ◽  
Ilya Frolov ◽  
Joseph Icenogle ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Nonstructural Protein ◽  
Relative Survival ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Minus Strand ◽  
Structural Protein ◽  
Coding Region ◽  
Reading Frame

ABSTRACT A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3′ cis-acting elements (3′ CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3′ CSE varied. The 3′ region necessary for optimal relative survival consisted of the 3′ 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3′ CSE. Within the 3′ CSE, deletions in the ∼245 nt that overlap the 3′ end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the ∼60-nt 3′ untranslated region (UTR) were lethal. None of the 3′ CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5′ proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3′ CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3′ CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3′ CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.

scholarly journals Development of a tRNA-dependent in vitro translation system

RNA ◽  
2001 ◽  
Vol 7 (5) ◽  
pp. 765-773 ◽  
Author(s):  
RICHARD J. JACKSON ◽  
SAWSAN NAPTHINE ◽  
IAN BRIERLEY
Keyword(s):  
In Vitro Translation ◽  
Translation System

scholarly journals Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

scholarly journals Arabidopsis Cell-free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures

2012 ◽  
Vol 53 (3) ◽  
pp. 602-602
Author(s):  
K. Murota ◽  
Y. Hagiwara-Komoda ◽  
K. Komoda ◽  
H. Onouchi ◽  
M. Ishikawa ◽  
...  
Keyword(s):  
Callus Cultures ◽  
In Vitro Translation ◽  
Translation System ◽  
Cell Free Extract ◽  
Arabidopsis Callus

scholarly journals An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

RNA ◽  
2008 ◽  
Vol 14 (3) ◽  
pp. 593-602 ◽  
Author(s):  
V. V. Zeenko ◽  
C. Wang ◽  
M. Majumder ◽  
A. A. Komar ◽  
M. D. Snider ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
In Vitro Translation ◽  
Translation System ◽  
Translational Inhibition ◽  
Eif2 Phosphorylation

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2767-2777 ◽  
Author(s):  
J.L. Villano ◽  
F.N. Katz
Keyword(s):  
Optic Lobe ◽  
Regional Growth ◽  
Positional Information ◽  
In Vitro Translation ◽  
Translation System ◽  
Regional Pattern ◽  
Microsomal Membranes ◽  
Position Sensitive ◽  
Coordination Function

Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs.

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

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