Evidence That the 32,000-Dalton Protein Encoded by Bottom-Component RNA of Cowpea Mosaic Virus is a Proteolytic Processing Enzyme

Translation of middle-component RNA of cowpea mosaic virus in vitro produced two polypeptides of 95 and 105 kilodaltons (95K and 105K, respectively) with overlapping amino acid sequences, which were specifically cleaved by a protease encoded by the bottom-component RNA. The proteolytic cleavage was studied by the addition of antibodies raised against various bottom-component RNA-encoded proteins to extracts prepared from bottom-component RNA-inoculated cowpea protoplasts. Since antiserum to the 32K polypeptide efficiently inhibited the proteolytic activity of such extracts, although antiserum to VPg or to the 170K polypeptide did not, evidence was obtained which indicates that the 32K polypeptide represents the protease involved. Fractionation of proteolytically active extract by glycerol gradient centrifugation demonstrated that 32K polypeptides do not exist as free proteins but are aggregated to the bottom-component RNA-encoded 170K, 84K, 60K, or 58K polypeptides. Maximal proteolytic activity was observed for 32K polypeptides associated with 170K polypeptides, suggesting that the activity was unstable and confined to newly synthesized molecules.     

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

Feline calicivirus (FCV), a member of the Caliciviridae, 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 for trans 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.     

Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses

Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).     

A retinoic acid-responsive gene, MK, found in the teratocarcinoma system. Heterogeneity of the transcript and the nature of the translation product

A newly identified gene, MK, is transiently expressed in the early stages of embryonal carcinoma cell differentiation and in the mid-gestation period of mouse embryogenesis (Kadomatsu, K., Tomomura, M., and Muramatsu, T. (1988) Biochem. Biophys. Res. Commun. 151, 1312-1318). Analysis of various MK cDNA clones revealed differences in the 5'-region. So far three classes of cDNA clones (MK1, MK2, and MK3) have been identified; they were different in the 5'-untranslated region but shared the rest of the sequence. Ribonuclease protection, RNA blotting, and primer extension revealed that MK2-type RNA was the major MK RNA in retinoic acid-treated embryonal carcinoma cells. In addition, the number of A residues in an oligo(A) stretch in the 5'-side of the common sequence differed from 9 to 29. The number was 9 in the most frequent cases, when the putative MK polypeptide had a molecular weight of about 15,500 and had a signal peptide-like sequence. Hybrid selected MK RNA yielded the predicted polypeptide upon in vitro translation. When pancreatic microsomal membranes were included in the translation system, the translation product of MK RNA was processed and entered into the lumen of the membranes. These results suggest that the product of the MK gene is an extracellular polypeptide.     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

The translation products from Cowpea Mosaic Virus (CPMV) RNAs obtained in two different cell-free systems were compared with the viral polypeptides synthesized in CPMV-infected cowpea protoplasts. It was shown that in both the wheat germ system and the rabbit reticulocyte lysate CPMV M component RNA was translated into two polypeptides of 105,000 and 95,000 dalton, which were not detected in CPMV-infected protoplasts. B component RNA however, gave different products depending on the system used. In the reticulocyte system this RNA was translated into a 200,000 dalton polypeptide which was further cleaved to give 170,000 and 32,000 dalton polypeptides. In the wheat germ system this processing step was lacking as only the 200,000 dalton product was formed. Since the 170,000 and 32,000 dalton polypeptides were also found in CPMV-infected protoplasts the two in vitro systems used apparently represent different stages of the expression of the B component RNA, thus providing a tool to study the mechanism of CPMV gene expression in vivo.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3.

We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.     

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.     

Orientation of the cleavage map of the 200-kilodalton polypeptide encoded by the bottom-component RNA of cowpea mosaic virus

The genomic organization of the bottom-component RNA of cowpea mosaic virus was studied. In vivo, this RNA encodes at least eight different polypeptides of 170, 110, 87, 84, 60, 58, 32, and 4 kilodaltons (K), the last polypeptide representing the genome-bound protein VPg. In rabbit reticulocyte lysates, bottom-component RNA is translated into a 200K polypeptide which is then processed to give the 32 and 170K polypeptides also found in vivo. By pulse-labeling the 200K primary translation product, we now show that the 32 and 170K polypeptides are derived from the NH₂-terminal and COOH-terminal parts of this polypeptide, respectively. Comparison of the proteolytic peptide patterns of 170K polypeptides synthesized in vitro and pulse-labeled at either the NH₂-terminal or the COOH-terminal end with the patterns of the 170 and 110K polypeptides found in vivo demonstrates that the order within the 200K primary translation product of cowpea mosaic virus bottom-component RNA is as follows: NH₂-32K polypeptide-58K polypeptide-VPg-24K polypeptide-87K polypeptide-COOH.     

Processing Pisum sativum seed storage protein precursors in vitro

The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.