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.     

The coat protein genes of squash mosaic virus: cloning,sequence analysis,and expression in tobacco protoplasts

Summary Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3 end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.     

Synthesis in vitro of the coat protein of papaya mosaic virus

Papaya mosaic virus (PMV) RNA directs the synthesis in vitro in either the wheat germ or reticulocyte lysate translation systems of numerous products. Three polypeptides are most prominent: pA (155,000 molecular weight), pB (73,000), and pC (22,000). The last, pC, displays the same molecular weight as coat protein and is precipitated by antibodies raised against PMV. The identities of pA and pB are less clear, although peptide mapping suggests that they and many of the minor products of translation are related to each other, but not to pC. The ability of partially encapsidated PMV ribonucleoprotein particles to direct protein synthesis has been assessed. As the extent of encapsidation increased, the relative synthesis of pA and pB decreased markedly. In contrast, the synthesis of pC persisted until a substantial fraction of the PMV RNA was completely encapsidated. In view of the polarity of assembly of PMV in vitro (M. Abouhaidar and J. B. Bancroft, (1978), Virology 90, 54-59) this finding indicates that the coding sequences for PMV coat protein are localized toward the 3' end of PMV RNA. A kinetic analysis of coat protein synthesis, coupled with Northern blot analysis of PMV RNA during translation suggest that PMV RNA must be cleaved to activate it for in vitro coat protein synthesis.     

Translation of red clover necrotic mosaic virus RNA in rabbit reticulocyte lysate: identification of the virus coat protein cistron on the larger RNA strand of the bipartite genome

The bipartite genome of red clover necrotic mosaic virus (RCNMV) was translated in nuclease-treated rabbit reticulocyte lysate with the synthesis of three principal translation products of molecular weights 39,000, 36,000, and 34,000. The 39,000 translation product was shown to be RCNMV coat protein by peptide mapping after partial proteolysis and by specific immunoprecipitation. RCNMV coat protein was shown to be coded for on the larger RNA (RNA-1) of the bipartite genome by hybrid-arrested translation using complementary DNA prepared to each of the fractionated RNAS. The 36,000-molecular weight product was also translated from RNA-1 while the 34,000-molecular weight product was not. Preliminary evidence indicated that the RCNMV coat protein is translated from a subgenomic species of RNA-1.     

Translation of soybean mosaic virus RNA in vitro: evidence of protein processing

The genomic RNA of soybean mosaic virus (SMV), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. The RNA was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. Immunological tests and peptide analyses indicate that six translation products are related to SMV coat protein, and one of these comigrated with coat protein during electrophoresis. Two other antigenically distinct classes of polypeptides were identified by their specific immunoprecipitation with antibody against the SMV cytoplasmic inclusion body protein or tobacco etch virus nuclear inclusion body protein. To determine if any products of the in vitro translation reactions resulted from proteolytic processing of a precursor molecule, translation reactions were carried out with amino terminal label N-formyl[35S]methionyl-tRNA(Met)i (f-Met), or with [35S]methionine, and the resultant products were compared. The putative SMV coat protein and a translation product related to the nuclear inclusion body protein were not labeled with f-Met indicating that they were generated by proteolytic processing at their amino termini. Consistent with this finding is the accumulation of new polypeptides of greater apparent molecular weight when amino acid analogs were present during translation.     

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.     

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 of soil-borne wheat mosaic virus RNAs

The genome of soil-borne wheat mosaic (SBWMV) virus appears to be composed of two RNAs. Three strains have the same large RNA, designated 1.0L RNA, but differ in the size of the smaller RNA, designated 0.5L RNA for wild type (WT), 0.4L RNA for mutant Lab 2, and 0.35L RNA for mutant Lab 1, where L corresponds to approximately 6700 nucleotide residues. The major translation products of 1.0L RNA in rabbit reticulocyte lysates had apparent molecular weights of 180,000 (180K), 152K, 135K, 80K, and 45K. None of these were precipitated with antiserum against virions. The 0.5L RNA stimulated the synthesis of products of 90K, 28K, and 19.7K, the 0.4L RNA of 66K, 28K, and 19.7K, the 0.35L RNA of 55K, 28K, and 19.7K. Protein of 19.7K comigrated with viral coat protein and was the predominant product in all cases. Immunoprecipitation, peptide mapping, and the time course of appearance of products suggest that the larger products of RNA Its (0.5L, 0.4L, and 0.35L RNA) arise from readthrough. The pattern of products is consistent with formation of 0.4L and 0.35L by internal deletions in the 3' region of 0.5L RNA. Extracts of SBWMV-WT-infected wheat contained polypeptides that corresponded to the translation products of 0.5L RNA in electrophoretic mobility and immunological reactivity.     

Translational strategies in potexviruses: products encoded by clover yellow mosaic virus, foxtail mosaic virus, and viola mottle virus RNAs in vitro

We have examined the template properties of the genomic RNAs from three potexviruses: clover yellow mosaic virus (CYMV), foxtail mosaic virus (FMV), and viola mottle virus (VMV). All three RNAs encode a large (160,000 to 182,000 molecular weight) nonstructural protein when translated in the rabbit reticulocyte cell-free system. Only CYMV RNA is able to direct the synthesis of significant quantities of a product whose electrophoretic mobility, antigenic determinants, and partial peptide map resemble those of authentic coat protein. All three viral RNAs also encode a number of discrete polypeptide products whose molecular weights are intermediate between those of the large nonstructural proteins and those of their respective coat proteins. We have examined the kinetics of synthesis in vitro of these intermediate products. For each of the three viral templates, as well as for papaya mosaic virus RNA, pulse-chase experiments suggest that most, if not all, the intermediate polypeptides are incomplete ("paused") or prematurely terminated precursors to the corresponding large nonstructural protein. A partial proteolytic map of the in vitro products encoded by CYMV RNA supports this interpretation. Our data, and those of others, are consistent with a model in which all potexviruses would encode a large nonstructural protein.     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

The molecular basis of the antigenic cross-reactivity between measles and cowpea mosaic viruses

Two nonrelated viruses, cowpea mosaic virus (wtCPMV) and measles virus (MV), were found to induce cross-reactive antibodies. The nature of this cross-reactivity was studied and results are presented here demonstrating that antiserum raised against wtCPMV reacted with peptide from the fusion (F) protein of MV. Furthermore, the F protein of MV was shown to share an identical conformational B cell epitope with the small subunit of CPMV coat protein. Passive transfer of anti-wtCPMV antibodies into BALB/c mice conferred partial protection against measles virus induced encephalitis. The results are discussed in the context of cross-protection.     

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.     

Nonstructural alfalfa mosaic virus RNA-coded proteins present in tobacco leaf tissue

The proteins synthesized under the direction of alfalfa mosaic virus RNAS in tobacco leaves have been examined under conditions of suppressed host protein synthesis. Besides the coat protein we could detect a 22K (K = apparent molecular weight in thousands), a 35K, and a set of 54K proteins. The 22K protein is serologically related to the coat protein. The 35K protein comigrated with the 35K protein whose synthesis is directed by RNA 3 in vitro The 54K proteins are serologically related to the 35K protein produced in vitro. Readthrough products of the 35K protein cistron into the coat protein cistron have been found previously in wheat germ extracts programmed with RNA 3. Two of these proteins comigrate with the 54K proteins synthesized in vivo. Since the 35K and the coat protein cistrons are read in different reading frames the formation of readthrough products is puzzling. In viruses with a tripartite genome the subgenomic mRNA for coat protein, RNA 4, is not known to be replicated as a separate genome entity. This might indicate that proteins synthesized by readthrough into the coat protein cistron play an essential role during replication, especially in the earliest phases.     

Partial characterization of two serologically related DNA-binding proteins specified by the cottontail rabbit herpesvirus CTHV

Summary We have previously described two serologically related DNA-binding phosphoproteins of different apparent molecular mass (75 kDa and 35 kDa) produced in cottontail rabbit herpesvirus (CTHV)-infected cells. The 75 kDa protein appeared before the 35 kDa protein in the infectious cycle. Here, we extend the characterization of these proteins. Protease V8 fingerprints of methionine-labelled 35 kDa protein showed four major peptide products, three of which comigrated with major peptides from digests of the 75 kDa protein. The fourth peptide, with an estimated mass of 10 kDa, reacted with an antiserum recognizing both proteins. In vitro translation of total or poly A-containing RNA isolated from infected cells at 24 h to 72 h post-infection produced only the 75 kDa protein as measured by immunoprecipitation with anti-75/35 kDa serum, suggesting that the 35 kDa protein is derived from the 75 kDa protein by proteolytic cleavage. Virus-specific RNA obtained by prehybridization to CTHV DNA also produced the 75 kDA protein, confirming its viral origin. The putative gene for the 75 kDa protein was localized to a region on the CTHV DNA restricted byPvuII.     

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     

Translation of turnip rosette virus RNA in rabbit reticulocyte lysates

Turnip rosette virus (TRosV) RNA was translated in nuclease-treated rabbit reticulocyte lysates, and four principal products (radioactive proteins always found under routine conditions) with molecular weights of 105,000, 67,000, 35,000, and 30,000 were synthesized. Optimum conditions for translation included 50 mM added K+, no added Mg2+, TRosV RNA at 25 to 50 mug/ml, incubation for 40 to 60 min; these conditions gave 20-fold stimulation in the incorporation of [35S]methionine over endogenous background levels. The 30,000 MW protein was identified as virus capsid protein by peptide mapping of specific immunoprecipitates. Most efficient synthesis of the capsid protein was observed for a virus-encapsidated subgenomic RNA (denatured MW 0.5 x 10(6)). The major genome products of 105,000, 67,000, and 35,000 MW were shown to be related by peptide analysis following partial proteolysis. Time course analyses suggested that the 67,000 product was processed from the 105,000 product by post-translational cleavage. The protease activity responsible for product cleavage was apparently viral coded and protease inhibitor studies further indicated the enzyme was of the thiol type. In an initiation assay using anisomycin, two ribosomes bound per TRosV RNA. The strategy of translation as determined for the virus here is discussed.     

Translation of tobacco rattle virus RNA in vitro using wheat germ extracts.

When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.