Mapping of the two coat protein genes on the middle RNA component of squash mosaic virus (comovirus group)

Squash mosaic virus, a member of the comovirus group, has a divided genome identified as middle-component RNA (M RNA; MW = 1.4 x 10(6)) and bottom-component RNA (B RNA; MW = 2 x 108). The isometric capsid of squash mosaic virus is composed of two distinct protein monomers with molecular weights of 22,000 (22k) and 42k. The isolated RNA components were translated in a rabbit reticulocyte lysate system. Translation products of the B RNA had estimated molecular weights of 190k, 51k, and 32k, while the M RNA products ranged in estimated molecular weight from 22k to 112k. Products of the B RNA did not react with antisera prepared to the 22k and 42k coat proteins. All of the M RNA products reacted with antiserum to the 22k coat protein and all the products larger than 35k reacted with antiserum to the 42k coat protein. The 22k product of M RNA translation had a V-8 protease peptide pattern identical to that of the 22k coat protein. Protease peptide patterns of the M RNA 64k and 112k-105k translation products showed a number of peptide fragments similar to those produced by the 22k and 42k coat proteins, indicating that the translation products contained the sequences of the two coat proteins. The proposed gene order of translation for squash mosaic virus M RNA is as follows: 5' end-22k coat protein gene-42k coat protein gene-48k unidentified protein gene-untranslated sequence-3' end.     

Coat protein and protease activity as in vitro translation products of potato carlavirus M RNA

Genomic-size RNA isolated from purified potato carlavirus M (PVM) was translated in both the reticulocyte and the wheat germ cell-free, messenger-dependent systems. The PVM RNA translated the same set of major products in both in vitro systems. The Mr values of the most prominent polypeptides observed consistently were 185,000 (P185), 147,000 (P147), 94,000 (P94), 87,000 (P87), 72,000 (P72), 67,000 (P67), 52,000 (P52), 46,000 (P46), 35,000 (P35) and 25,000 (P25). Relatively low amounts of a translation product of Mr 200,000 (P200) were often detectable in both systems. The P35 polypeptide displayed the same molecular weight and one-dimensional peptide map as the virus coat protein (CP), and was precipitated by antibodies raised against PVM and PVM CP. The kinetics of appearance of the in vitro synthesized polypeptides suggested that primary translation products of high molecular weight undergo post-translational proteolytic cleavage.     

Translation in vitro of carnation mottle virus RNA Regulatory function of the 3′-region

Carnation mottle virus (CarMV) RNA was translated in a wheat-germ, cell-free system into three discrete polypeptide chains of 77,000 (CP-I), 38,000 (CP-II) and 30,000 M_r (CPIII). The size of the three polypeptide products represents the translation of almost the entire length of the viral RNA. The CP-II in vitro product was identified as the viral coat protein by gel electrophoresis under denaturing conditions, inununoprecipitation with antibodies directed against the disrupted viral particles, and by peptide mapping. The discrete nature of the three proteins was shown by peptide analysis of the proteolytic products, the absence of cross-reaction between antibodies against CarMV-disrupted particles and CP-I or possibly CP-III, and by the order of their appearance during in vitro translation. Limited phosphorolysis of the 3′-terminus of the viral RNA chains by Escherichia coli polynucleotide phosphorylase caused the selective disappearance of the 77,000 protein product, paralleled with a gradual loss of infectivity. These observations might indicate that the viral RNA is polycistronic, and suggest that the structure at the 3′-terminus of the carnation mottle virus RNA may have a regulatory role in the translation of the viral RNA chains.     

Analysis of alfalfa mosaic virus 17 S RNA translational products

In a previous paper, the problem has been raised whether or not the coat protein gene of alfalfa mosaic virus, although present on the 17 S RNA, is translated in a cell-free system derived from Krebs-II ascites cells. The translation products of 17 S RNA have been separated by electrophoresis on polyacrylamide gels and the tryptic peptides analyzed by fingerprinting. The results show that the only in vitro translation product of 17 S RNA consists of a 35,000 MW polypeptide quite different from the coat protein, which consequently is not translated. This suggests that, in contrast to the other AMV RNA's the 17 S is only partially translated.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes

Cucumber mosaic virus (CMV) contains four major RNA species, designated RNAs 14 in order of decreasing molecular weight. As a preliminary means of analysing gene content and distribution between these RNA species, each of the RNAs was purified and used to direct protein synthesis in cell-free systems from wheat embryos, wheat germ, and rabbit reticulocytes, and in intact oocytes of the toad, Bufo marinus. In vitro translation products of the CMV RNAs (Q strain) were analysed by electrophoresis on discontinuous slab polyacrylamide gels in the presence of sodium dodecyl sulphate. RNA 1 and RNA 2 proved untranslatable in oocytes but both directed synthesis of a large number of products in the wheat and rabbit reticulocyte cell-free systems. The highest molecular weight products obtained, about 105,000 for RNA 1 and 120,000 for RNA 2, represent most of the coding potential of these RNAs. Thus, RNA 1 and RNA 2 may be monocistronic messengers for the 105,000 and 120,000 MW products, respectively. Translation of RNA 3 in the wheat cell-free systems yielded three major products of molecular weights 34,000–39,000. In contrast, RNA 3 directed synthesis of essentially one product of molecular weight about 34,000 in both the rabbit reticulocyte system and oocytes. RNA 3 did not direct synthesis of coat protein in any of the systems, contrary to our earlier results and the known presence of the coat protein gene on RNA 3. RNA 4 directed synthesis of coat protein and higher- and lower molecular weight minor products in the cell-free systems and in oocytes. A product corresponding to the entire coding potential of RNA 4 was observed in the rabbit reticulocyte system, but not in the wheat systems or toad oocytes. The conflicting results obtained for translation of RNA 3 and RNA 4 in the different systems illustrate the danger in analysing genetic material by in vitro translation. The discussion considers which of the in vitro products are most likely to be synthesized in vivo and a tentative model of the CMV genome is proposed.     

Cell-free translation ofDrosophila C virus RNA: Identification of a virus protease activity involved in capsid protein synthesis and further studies onin vitro processing of Cricket paralysis virus specified proteins

Summary Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsids precursors of either of the viruses.With 7 Figures     

Highly purified cucumber mosaic virus-induced RNA-dependent RNA polymerase does not contain any of the full length translation products of the genomic RNAs

The extensively purified RNA-dependent RNA polymerase (RNA replicase) induced by infection of cucumber seedlings with cucumber mosaic virus (CMV) was investigated to determine whether the enzyme contains full-length translation products of the CMV genomal RNAs. Two experimental approaches were used. (1) RNA replicase preparations from plants infected with three strains of CMV (Q, P, and T) all had almost identical polypeptide compositions, which included a major polypeptide of relative mobility on sodium dodecyl sulfate-polyacrylamide gels of Mr, 100,000, and two other proteins, Mr 110,000 and Mr, 35,000, present in smaller, varying amounts. These polypeptides were unique to enzyme preparations from CMV-infected plants and had similar electrophoretic mobilities to the translation products of Q-CMV RNAs 1, 2, and 3, respectively. Analysis of the in vitro translation products of the corresponding RNAs of the other two strains of CMV showed, however, that those of P-CMV RNA 2 and T-CMV RNAs 1 and 3 varied significantly in size from the translation products of the corresponding Q-CMV RNAs. (2) Peptide mapping studies, using digestion with the V8 protease from Staphylococcus aureus or cleavage with CNBr, confirmed that none of these three components of the highly purified RNA replicase was a translation product of the CMV RNAs. The work reported in this paper, therefore, showed that the full-length translation products of the genomal RNAs of CMV were absent from the RNA replicase induced by this virus after solubilization and extensive purification.     

Evidence for an autoprotease activity of sindbis virus capsid protein

Sindbis virus capsid protein is virtually the only product formed when viral 26 S RNA is added to a mouse Krebs ascites cell-free protein synthesis system. However, substitution of arginine and proline by the respective analogues canavanine and azetidine-2-carboxylate inhibits capsid production and larger polypeptides accumulate. The latter are converted to capsid in pulse-chase experiments when the normal amino acids are added during the chase, but not if the chase period contains only the analogues in the reaction mixture. These results support an autoprotease model for the co-translational cleavage of Sindbis virus capsid proteins.     

A comparative study on the cell-free translation of the genomic RNAs of two aphid picorna-like viruses

Summary The genomic RNAs of aphid lethal paralysis virus (ALPV) andRhopalosiphum padi virus (RhPV)—two distinct picorna-like viruses found in aphids [Williamson et al. (1988) J Gen Virol 69: 787–795]—were both efficiently translated in rabbit reticulocyte lysates. ALPV RNA was translated into primary products with molecular weights ranging from 92 kDa to 170 kDa. These underwent time-dependent post-translational cleavage to produce smaller polypeptides including some with molecular weights comparable to those of the viral structural proteins. A 92 kDa polypeptide as well as smaller proteins were immunoprecipitated with capsid protein antisera, indicating the presence of at least one large capsid subunit protein precursor. RhPV RNA was translated into products of molecular weights ranging from 45 kDa to 175 kDa. There was no evidence for time-dependent post-translation cleavage of RhPV translation products. However, a 60 kDa polypeptide was precipitated with antiserum to RhPV virions, indicating that at least one capsid protein of RhPV is derived by proteolysis of a precursor protein, like those of ALPV and most other picornaviruses.     

A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates has been used to analyse the pathway of proteolytic processing of the primary translation products. A minimum of two distinct proteases is required to account for the results: one for the excision of the capsid precursor protein, A1, from the nascent polyprotein, and the other for all other cleavages including cleavage at the F/C junction. The excision of A1 is an extremely rapid reaction which occurs as soon as the cleavage site has been synthesised and is resistant to all the proteolytic inhibitors tested and to high temperature, characteristics which are more consistent with an intramolecular cleavage catalysed by a virus-coded protease than cleavage by an endogenous reticulocyte protease. Once excised, A1 remains stable until translation has reached the middle of the region of the genome coding for C, at which time a number of events occur in rapid succession: F is excised in its mature form probably via an intramolecular cleavage; a proteolytic activity capable of secondary processing of A1 to A, B, D1, alpha, gamma and epsilon appears; and a polypeptide of molecular weight about 32,000 appears. This protein (p32) originates from the N-terminal portion of C, and maps in the same position on the genome as p22, the protein previously identified as the virus-coded protease. Polypeptide p32 is derived from C by a single step cleavage generating E as the other product, a processing pathway at least as important, if not more important than the step-wise route via D as an intermediate. Since p32 first appeared at the same time as the start of secondary processing, whilst p22 was first detected much later, it is argued that at least the early stages of processing of the capsid precursor must have been carried out by p32 rather than p22.     

Nucleotide sequence and translation of satellite tobacco mosaic virus RNA

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [gamma-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5' termini of encapsidated STMV RNAs were not phosphorylated. The 240 3'-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5'-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5'-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5'-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5' and 3' termini, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

The synthesis and processing of the nepovirus grapevine fanleaf virus proteins in rabbit reticulocyte lysate

Translation of a mixture of the two grapevine fanleaf virus (GFLV) RNAS in reticulocyte lysates in the presence of the amino acid analogs canavanine, S-aminoethyl-cysteine, and p-fluorophenylalanine gave two products of molecular weights (Mr) 220,000 (from RNA-1) and 125,000 (from RNA-2). In the absence of these analogs a protease apparently induced by RNA-1 catalyzed cleavage of the RNA-2 product (Mr 125,000) into two proteins of Mr 68,000 and 58,000. The 58,000 Mr protein was shown to be the likely virus coat protein by peptide mapping after partial proteolysis. Cleavage of the RNA-2 product of GFLV was not catalyzed by the translation products of another nepovirus, tobacco ringspot virus.     

Expression of 67,000 MW calcimedin and its binding protein in resident and thioglycolate-elicited macrophages.

Calcimedins are newly recognized calcium-dependent hydrophobic-binding proteins. The 67,000 MW calcimedin exhibits calcium-dependent specific binding to a 60,000 MW intracellular acceptor protein (60,000 MW). Macrophages possessed 30,000 MW and 67,000 MW calcimedins and 60,000 MW acceptor protein, identified by immunofluorescence and confirmed by SDS-PAGE and Western immunoblots. Compared with low levels in resident macrophages, thioglycolate-elicited inflammatory macrophages showed large increases in the levels of 67,000 MW calcimedin and 60,000 MW acceptor protein, especially at cell-limiting membranes. Levels of calmodulin and 30,000 MW calcimedin appeared similar in both populations. Trypsinization removed most 67,000 MW calcimedin and some 60,000 MW acceptor-protein staining. The calcimedins appear to represent novel macrophage-differentiation antigens. Their modulation suggests that they may play a role in the inflammatory response.     

Translation of coxsackievirus B RNAs in a rabbit reticulocyte lysate: Characterization of the genome RNA,reaction conditions for translation,and analysis of the products

Summary The genomic RNA of coxsackievirus B5 was characterized, and the RNAs from B4 and B5 viruses were translated in a micrococcal nuclease-treated rabbit reticulocyte lysate cell-free system to characterize the products. The viral RNA, native or denatured, sedimented as a sharp symmetrical peak between 45S and 28S marker rRNAs; its estimated molecular weight was 2.53×10⁶. It was polyadenylated and contained about 7,440 nucleotides which could code for proteins with a total molecular weight of approximately 273,000. At saturating concentrations a five- to sevenfold stimulation in methionine incorporation over background was obtained when translating either B4 or B5 RNA. The translation directed by RNA was inhibited by pactamycin, aurintricarboxylic acid, puromycin, or cycloheximide (inhibitors of initiation and elongation) and by RNase. 7-Methylguanosine-5-monophosphate, a cap analog which inhibits protein synthesis directed by capped messengers, did not inhibit this translation, suggesting that B5 RNA may not be capped. At least 10 polypeptides with apparent molecular weights of 31,000 to 98,000 were synthesized in the presence of either RNA; many of these comigrated with polypeptides synthesized in the virus-infected HeLa cells. A polypeptide (MW 36,000) of B4 RNA-directed products, which comigrated with a similar one from the virus-infected cells, also comigrated with an authentic capsid protein VP2 of the virus. The products of B4 RNA-directed synthesis were different from B5 RNA-directed products: the former contained at least five polypeptides of MW 31,000 to 45,000, while the latter contained only one (31,000). Antiserum to B5 virus capsid proteins precipitated several cell-free polypeptides with molecular weights of 52,000 to 92,000, suggesting that an immunologic relationship may exist between cell-free products and the capsid proteins. Comparison of partial proteolytic digests of cell-free products with molecular weights of 69,000 to 92,000 and authentic capsid proteins also suggests strongly that the products may contain some sequences of B5 virus capsid proteins.With 5 Figures     

Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration.     

Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli

We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease.     

Translation of potyvirus RNA in a rabbit reticulocyte lysate: Cell-free translation strategy and a genetic map of the potyviral genome

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) in a rabbit reticulocyte lysate were analyzed to determine the effect of RNA quality on template activity and to identify all the products of the potyviral genome. The size distribution of the in vitro translation products, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was dependent on RNA quality. Full-length RNA stimulated the cell-free synthesis primarily of a 87,000 (87K) molecular weight product for TEV and a 78K product for PeMV. RNA with limited fragmentation stimulated a number of discrete products, while highly fragmented RNA did not act as a template. RNA monocistronic for capsid protein was not detected. Four of the six discrete translation products were identified by reactivity with antisera to four virus-specific proteins. A number of other products, which were consistently immunoprecipitated with more than one antiserum, were detected. Products were presumed to be premature terminations and/or gene readthroughs on the basis of serological reactions and of molecular weight estimates. These readthroughs were useful in linking genes and in constructing a genetic map of the potyviral genome. The proposed genetic map for TEV is as follows: 5' end-87K protein gene-49K nuclear inclusion protein gene-50K protein gene-70K cylindrical inclusion protein gene-54K nuclear inclusion protein gene-30K capsid protein gene-3' end. The proposed genetic map for PeMV is as follows: 5' end-78K protein gene-49K protein gene-41K protein gene-68K cylindrical inclusion protein gene-56K protein gene-33K capsid protein gene-3' end. The proposed genetic map accounts for 95% of the estimated coding capacity of the TEV RNA and 93% of the PeMV RNA.     

Translation of potyvirus RNA in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) RNA in the mRNA-dependent rabbit reticulocyte lysate produced products which comigrated with potyviral inclusion proteins on polyacrylamide gels. The TEV RNA translation products comigrating with the 49,000 and 54,000 molecular weight TEV nuclear inclusion proteins were also immunoprecipitated by antisera specific to the nuclear inclusion proteins. The proteolytic peptide map of the comigrating in vitro translation products for TEV RNA was similar to that generated by the nuclear inclusion proteins. The PeMV RNA translation product comigrating with the cylindrical (pinwheel) inclusion protein was immunoprecipitated by antiserum to PeMV cylindrical inclusion protein. These results provided direct evidence that the inclusions associated with potyviruses consist of virus-coded, nonstructural proteins.