Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase

Mouse L cells that lacked thymidine kinase (L_tk^? cells) were infected with wild-type vaccinia virus or with a mutant that was deficient in the viral thymidine kinase. Cytoplasmic messenger RNA preparations were isolated 2 hr after infection and translated in a messenger RNA-dependent reticulocyte lysate. Both messenger RNA preparations directed the efficient synthesis of early vaccinia virus polypeptides. Furthermore, translation of the messenger RNA from cells infected with wild-type virus resulted in a substantial increase in the thymidine kinase activity of the lysate which was prevented by inhibitors of cell-free protein synthesis. Translation of the messenger RNA from cells infected with the viral tk^? mutant, or from uninfected L_tk^? cells, did not increase the thymidine kinase activity of the extract. The enzyme synthesized in vitro was compared with that isolated from vaccinia virus-infected cells. The two enzymes were indistinguishable with respect to their rates of thermal inactivation, their sedimentation coefficients in glycerol gradients, and their relative electrophoretic mobilities in nondenaturing polyacrylamide gels. We conclude that active viral thymidine kinase can be synthesized de novo in a cell-free system directed by early messenger RNA from vaccinia virus-infected cells.     

Cell-free translation of carnation latent virus RNA and analysis of virus-specific dsRNA

Carnation latent virus was shown to direct the synthesis of virus-specific polypeptides in both reticulocyte lysate and wheat germ in vitro translation systems. The L-(4,5-³H)-leucine-labeled products ranged in molecular mass from M_r 190 to 33 kD. The 33 kD product, synthesized after only 15 min incubation, was the only major polypeptide that immunoprecipitated with antiserum to CarLV. Coatprotein synthesis does not occur as a result of proteolytic processing, but may arise as a result of translation of a subgenomic RNA species. Subgenomic RNA species were not detected by Northern hybridization of CarLV cDNA to either viral RNA or total nucleic acid from systemically infected plants, although CarLV-specific dsRNA species equivalent to 1.6 and 2.1 kb were detected.     

Differential precipitation of foot and mouth disease virus proteins made in vivo and in vitro by hyperimmune and virus particle guinea pig antisera

Foot-and-mouth disease virus (FMDV)-specified proteins synthesized in infected cells or made in reticulocyte lysates from FMDV RNA have been reacted with hyperimmune or virus particle antiserum and the immune complexes precipitated using protein A bearing Staphylococcus aureus ghosts. Hyperimmune serum precipitates most of the virus-specific proteins efficiently but some of the small polypeptides and P52 and P34, polypeptides derived from the middle of the genome, are precipitated poorly or not at all. Virus particle antiserum precipitates predominantly virus structural proteins and their precursors. Immunoprecipitation of in vitro lysates primed by type A FMDV RNA with type O antisera indicates that some antigenic determinants are shared by polypeptides of the two serotypes. Precipitation of the products of translation of type O virus RNA in vitro shows that the FMDV genome can be translated completely in a reticulocyte lysate and the products efficiently processed.     

Selective inhibition of protein synthesis by synthetic and vaccinia virus-core synthesized poly(riboadenylic acids)

Studies were undertaken to compare the effect of poly(A)s from various sources on selective inhibition of protein synthesis in the reticulocyte lysate system programmed with viral and cellular mRNAs. RNA synthesized in vitro by vaccinia virus cores in the presence of only ATP inhibited overall HeLa cell polypeptide synthesis by over 80% with a minimal effect on translation of vaccinia virus mRNAs. Hybridization of the [alpha-32P]AMP-labeled RNA made in vitro by vaccinia virus cores in the presence of only ATP, showed no complementary to HindIII restriction fragments of vaccinia virus DNA indicating that the in vitro product was poly(A). Fractionation of synthetic and core-synthesized poly(A) into three size classes showed that the larger the size of poly(A), the greater its inhibitory activity of protein synthesis in the cell-free system. Inhibition of translation of mRNAs from vaccinia virus-infected HeLa cells was also observed in the presence of poly(A). However, virus-induced polypeptide synthesis was more resistant to the effect of poly(A) than were cellular polypeptides. Oligo(dT) when added to the reticulocyte lysate system was capable of reversing the inhibition of protein synthesis caused by both core-synthesized poly(A) and core-transcribed RNAs. These results indicate that poly(A) synthesized by the virion-associated enzyme has inhibitory properties similar to those of synthetic poly(A).     

Comparative analysis of polypeptides synthesized in vivo and in vitro by two strains of barley stripe mosaic virus

Proteins synthesized in a wheat germ system containing RNA from the Type or ND18 strain of barley stripe mosaic virus (BSMV), or polyribosomes from Type or ND18-infected barley, were compared with each other and with polypeptides from Type and ND18-infected plants. Polypeptides with apparent molecular weights (Mr) of 25, 67, and 120 x 10(3) that were induced in infected barley were synthesized in vitro by polyribosomes from infected plants or RNA from purified BSMV. The 25,000 Mr polypeptide was identified as BSMV coat protein by coelectrophoresis with coat protein from purified virions, by immunoprecipitation, and by absence of [35S]methionine incorporation into the protein. Strain-specific differences were observed in two additional translation products synthesized in both wheat germ and reticulocyte lysate systems containing Type of ND18 RNA. RNAs from both strains directed synthesis of 71 and 82 x 10(3) Mr polypeptides. However, ND18 RNA directed synthesis of a larger amount of the 71,000 Mr polypeptide, whereas with Type RNA the 82,000 Mr polypeptide predominated. These two proteins may be processed in vivo because reduced synthesis of both polypeptides in wheat germ extracts containing polyribosomes from infected plants was correlated with increased synthesis of the 67,000 Mr polypeptide. Several additional polypeptides synthesized in the wheat germ system were probably premature termination products because their synthesis in the reticulocyte lysate was greatly reduced.     

Translation of PVX RNA in vitro by wheat germ. I. Characterization of the reaction and product size

Potato virus X (PVX) RNA was an efficient mRNA for in vitro translation by the wheat germ system. Optimal incorporation of labeled amino acids occurred in reaction mixtures (50 μl) containing 3 mM Mg²⁺, 70 mM K⁺, and 3–4 μg of RNA. The presence of PVX RNA stimulated amino acid incorporation up to 40 times over background levels. Addition of spermine or spermidine further stimulated amino acid incorporation nearly twofold. PVX RNA translation was not inhibited by adding double-stranded RNA from Penicillium stoloniferum. The largest of several polypeptides obtained from PVX RNA translation reaction mixtures had an apparent molecular mass of 110,000 daltons. No polypeptide that coelectrophoresed with native PVX coat protein subunits was observed even when heat-treated PVX RNA was used, or when the products from spermine-stimulated reactions were analyzed.     

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 eggplant mosaic virus RNA in wheat germ extracts and reticulocyte lysates

Eggplant mosaic virus (EMV) RNA extracted from virions (virion EMV RNA) is a good messenger in wheat germ extracts and reticulocyte lysates but directs surprisingly little synthesis of coat protein. Large amounts of coat protein, however, are synthesized when a low molecular weight RNA purified from virion RNA is used as messenger. Heavy virion RNA directs the synthesis of two proteins of high molecular weight in reticulocyte lysates. The larger of these proteins accounts for almost the entire coding potential of 2 × 10⁶ dalton viral RNA.     

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982)Virology118, 401–410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun.71, 393–399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.     

Genetic characterization of plasmid formation by N- mutants of bacteriophage lambda.

Cells of plants infected with any of three strains of tobacco mosaic virus (the cowpea, common, and a wheat strain) contain, in addition to full-length virus rods, a heterodisperse population of rod fragments. RNA extracted from purified virus showed that some of these rods were of discrete sizes; the RNAs ranged in molecular weight from about 0.28 to 1.7 × 10⁶, containing 3′-ends identical to the original 3′-end of viral RNA, as judged by their capacities to bind a specific amino acid. Only the cowpea strain produced a rod containing a small (0.28 × 10⁶M_r) RNA, which is known to be the mRNA for coat protein. Each strain produced an RNA of 0.68 × 10⁶M_r (intermediate-length RNA) which coded in vitro (using a wheat germ system) for an ～30,000 M_r polypeptide. Other RNAs (0.9 to 1.7 × 10⁶M_r) were less similar in molecular weights among the three strains, but the predominant in vitro product of each was also the ～30,000 M_r polypeptide. peptide maps comparing the translation products of the short and intermediate RNAs of the cowpea strain showed they were distinct polypeptides. We conclude that at least some of the less-than-full-length viral rods found in preparations of the common and wheat strains of TMV, as well as those previously reported for the cowpea strain, represent the encapsidation of subgenomic portions of the viral RNA which are engendered during viral replication.     

The viral protein 3CD induces an equilibrium between the viral protein and RNA synthesis in a cell-free system for poliovirus replication

Summary.  In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log₁₀. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log₁₀ by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system. Received April 2, 2001 Accepted November 15, 2001     

In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus West Nile virus.

Virus-specific proteins synthesized in BHK cells infected with the flavivirus West Nile (WN) virus and in vitro using the virus-specific infectious 42 S plus-strand RNA as messenger RNA, have been studied. Mapping of the tryptic peptides indicates that the viral core protein V2 and the viral glycoprotein V3 do not share common sequences. No tryptic peptides identifiable by mapping were obtained from the viral membrane-associated protein V1. Seven apparently virus-specific intracellular proteins were detected by comparative SDS-PAGE of mock-infected and virus-infected [³⁵S]methionine-labeled cell lysates: p_i 15, p_i 20, p_i 27, p_i 37, p_i 49, p_i 71, and p_i 100 (the index i indicates the intracellular origin of the proteins, the number gives the apparent molecular weight in 10³ daltons). The proteins p_i 15 and p_i 49 represent the intracellular forms of the viral structural proteins V2 and V3, respectively. p_i 15 and V2 are not identical but differ slightly from each other. V1 has not been detected in infected cells. Mapping has shown that the other intracellular proteins (with the possible exception of p_i 37, which has not been analyzed) are unrelated to either V2 or V3 and do not share common sequences. They represent nonstructural proteins. The total molecular weight of the apparently unrelated nonstructural and structural proteins is about 290,000 daltons. Data obtained in a number of laboratories have shown that a virus-specific 42 S RNA molecule, which is structurally indistinguishable from the viral genome, probably functions as mRNA for all virus-specific proteins in vivo. This RNA has been isolated from WN virus-infected cells and translated in vitro in the wheat germ and the rabbit reticulocyte lysate system. Digestion of the total [³⁵S]methionine-labeled proteins synthesized in vitro in either of both systems with trypsin followed by peptide mapping has shown that the great majority of the resulting peptides are present in the structural proteins V2 and V3 and vice versa. No evidence was obtained for the in vitro synthesis of nonstructural proteins. Proteins synthesized in the reticulocyte lysate were fractionated by SDS-PAGE and isolated polypeptides studied by peptide mapping. Polypeptides of molecular weights between 11,500 and 90,000 daltons were obtained. Their peptide maps indicate that all polypeptides are translated from a single initiation sequence. The map of the smallest in vitro synthesized polypeptide P_retic 11.5, having a molecular weight of 11,500 daltons, was almost identical to that of V2. The map of the largest protein synthesized in significant amounts in vitro, the protein P_retic 90, was very similar to the map of a mixture of the viral proteins V2 and V3. The analyses suggest the following gene order on the 42 S RNA: 5′-terminus-V2-V3-(V1, p_i 20, p_i 27, p_i 37, p_i 71, p_i 100)-3′-terminus. The order of genes indicated in brackets remains to be determined. Some implications of these results concerning the possible mode of translation of flavivirus-specific 42 S RNA are discussed.     

Similar frequencies of antigenic variants in Sendai, vesicular stomatitis, and influenza A viruses

The effects of three inhibitors of glycosylation (2-deoxy-d-glucose, d-glucosamine, and tunicamycin) on the formation of infectious extracellular snowshoe hare virus and the synthesis of intracellular virus-induced polypeptides have been examined. The inhibitor 2-deoxy-d-glucose, used at concentrations of up to 10 mM, did not significantly affect either infectious virus release or viral-induced polypeptide synthesis and maturation. By contrast, both d-glucosamine (40 mM) and tunicamycin (1 μg/ml) prevented the release of infectious virus and inhibited the maturation of the two viral glycoproteins (Gl and G2), but not the viral nucleoprotein (N). Two new polypeptides, Gl₀ and G2₀, of molecular weights 105 × 103 and 33 × 103, respectively, were found in infected cells treated with tunicamycin or D-glucosamine. These two polypeptides were identified as virus specific since they could be precipitated by virus immune serum. Tunicamycin was found to inhibit the release of extracellular virus particles at 33 and 38°, which correlated with the observation that no intracellular virus particles were detected in electron micrographs of cells treated with the drug.     

Anticellular and antiviral effects of pppA(2'p5'A)n

pppA(2′p5′A)n, the 2′5′ linked oligoadenylic acid triphosphate commonly known as 2–5A, is synthesized by an enzyme fraction from interferon-treated cells and rabbit reticulocyte lysates. By the use of a calcium-phosphate coprecipitation technique, we show here that treatment of cells with 2–5A results in the inhibition of in vivo protein, RNA, and DNA synthesis. These inhibitory effects of 2–5A are transient at low concentrations of 2–5A (<20 nM). At higher concentrations, however, the inhibition of protein, RNA and DNA synthesis continues with an apparent effect on cell growth. Treatment of vesicular stomatitis virus (VSV)-infected cells with 2–5A results in the inhibition of virus RNA replication. In accord with this virus production is inhibited by 90–100% after treatment of infected cells with 10–100 nM of 2–5A. This inhibition seems to be mediated by the 2–5A-dependent nuclease which results in the degradation of viral RNA.     

In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells.

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [³⁵S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.     

The genome of equine arteritis virus.

Equine arteritis virus (EAV) contains an infectious RNA. [³H]uridine-labeled RNA was released from purified virus (density, 1.155 g/ml in sucrose; s_20,w, 224 ± 8 S) with sodium dodecyl sulfate and 2-mercaptoethanol. An s_20,w value of 48 S was found in isokinetic sucrose gradients in 0.1 M saline. In 1 mM saline, sedimentation was slower (33 S), in 0.1 M saline plus 1 mM MgCl₂, a value of 56 S was measured. A molecular weight of 4.0 × 10⁶ was determined by polyacrylamide-agarose-gel electrophoresis. Heating of purified RNA in the presence of 1.1 M formaldehyde and subsequent centrifugation in gradients containing formaldehyde did not result in degradation to smaller RNA's. From this procedure a molecular weight of 4.1 × 10⁶ was calculated. Buoyant density in Cs₂SO₄ of the RNA was 1.65 g/ml. In most experiments Semliki forest virus RNA was taken as a reference. It behaved almost indistinguishably from the RNA of EAV. In conclusion EAV contains an infectious, colinear molecule of single-stranded RNA with a molecular weight of about 4 million. These data justify a definite inclusion of this virus in the family Togaviridae.