Nature of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells.

Cytoplasmic and polyribosomal RNAs from Rous sarcoma virus-transformed and phenotypically reverted field vole cells were fractionated by rate-zonal sedimentation and hybridized with a (3)H-labeled complementary DNA viral probe to determine the size classes of virus-specific RNA present in these cell types. In contrast to Rous sarcoma virus-infected permissive avian cells, only two of three discrete species of virus-specific RNA were detected in the cytoplasm of these vole cells. These included genome-length 35S RNA and a 21S RNA. However, viral 28S RNA, routinely detected in the cytoplasm of productively infected avian cells, could not be found in cytoplasmic RNA from vole cells. In addition, a low-molecular-weight viral RNA sedimenting less than 16S was detected in both infected avian and vole cells. Because of its heterogeneity this latter species is most likely generated from the intracellular degradation of the larger viral RNAs. Both the viral 35S and 21S RNA were also found to be associated with total polyribosomes from these vole cells. Studies were also performed to determine the distribution of both total viral genomic and sarcoma-specific RNA sequences among the size classes of fractionated total polyribosomes. In both vole cell types the majority of cytoplasmic viral RNA sequences were also associated with polyribosomes and were similarly distributed among the size classes of total polyribosomes. Sarcoma-specific sequences were present on both the 35S and 21S RNA species. These data suggest that the expression of the viral transforming gene in revertant field vole cells may be controlled at some stage subsequent to translation of the viral RNA.     

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.     

Ability of translation factors from infected encephalomyocarditis viruses of ascites carcinoma cells to maintain the protein-synthetizing activity of cellular polyribosomes

A highly effective protein-synthesizing system fully dependent on translation factors was isolated from the ascite Krebs-2 carcinoma. The effect of EMC-infection on the ability of these factors to maintain the translation of polyribosomes containing cell mRNA was studied. It was shown that the activity of translation factors of infected cells does not differ from that of non-infected cells in terms of their ability to maintain the protein-synthesizing activity of cell polyribosomes in vitro.     

Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus.

RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.     

Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes

Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.     

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.     

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3''-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.     

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.     

Cyclic nucleotide-independent protein kinases bound to cytoplasmic and nuclear polyribosomes in non-infected and adenovirus-infected HeLa cells

Cyclic nucleotide-independent protein kinase activity bound to cytoplasmic and nuclear polyribsomes from non-infected and adenovirus-infected HeLa cells was compared. The enzymes catalysed the incorporation of phosphate from gamma-(32)P-labelled ATP or GTP into acid-precipitable material in the absence of exogenous substrates. Their activity was not affected by cyclic AMP or cyclic GMP and was not inhibited by a cyclic nucleotide-dependent protein kinase-inhibitor protein. The kinases are tightly bound to polyribosomes of either origin from infected and non-infected cells, since treatment with 0.5m-NaCl did not dissociate the activity. The enzymes and the enzyme-associated endogenous substrates of cytoplasmic polyribosomes are significantly different from those of the nucleus, and adenovirus infection of the cells did not alter the nature of the enzymes or the substrates at 18-20h after infection. Nuclear kinases catalysed 3-4-fold more phosphate incorporation than did the cytoplasmic kinases. They did not phosphorylate endogenous substrates in the cytoplasmic preparations, and vice versa, which suggests that such substrates for cytoplasmic and nuclear kinases are specific. Polyacrylamide-gel electrophoresis of the phosphorylated proteins revealed the presence of a higher number of endogenous substrates in the nuclear preparation. The nuclear kinases phosphorylated all histones from HeLa cells, but the cytoplasmic ones phosphorylated predominantly the histone of mol.wt. 12000. Bovine heart kinase phosphorylated several low-molecular-weight cytoplasmic proteins and no nuclear proteins. With a DEAE-cellulose column either enzyme activity could be resolved into a number of peaks. The substrate specificities of these peaks indicate that there are at least two different forms of the enzyme in each preparation of polyribosomes.     

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.     

Association of eukaryotic aminoacyl-tRNA-synthases with polyribosomes

Upon fractionation of a mitochondria-free extract of rabbit reticulocytes into a ribosome-free extract and mono- and polyribosomes the bulk of the aminoacyl-tRNA synthetase activity was found in the fraction of mono- and polyribosomes. All the fifteen aminoacyl-tRNA synthetases were revealed, although in somewhat different quantities, in both fractions of the mitochondria-free reticulocyte extract. Aminoacyl-tRNA synthetases of the ribosome-free extract are found in two forms: RNA-binding one, and, the one having no affinity for high molecular weight RNAs. Aminoacyl-tRNA synthetases dissociated from the complexes with polyribosomes exist only in the RNA-binding form. All aminoacyl-tRNA synthetases can be removed from such complexes by an addition of 16S rRNA of E. coli, poly(U) or tRNA of rabbit reticulocytes. This testifies to labile association of aminoacyl-tRNA synthetases with the RNA-component of polyribosomes as well as to a rather nonspecific character of their interaction. After EDTA-induced dissociation of polyribosomes, the aminoacyl-tRNA synthetase activity was detected in the complex with both ribosomal subunits.     

Transport of glutamine by rat kidney brush-border membrane vesicles.

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.     

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.     

Life history of the bird cherry-oat aphid, Rhopalosiphum padi, on transgenic and non-transformed wheat challenged with Wheat streak mosaic virus

The life history of the bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), was studied via laboratory assays on Wheat streak mosaic virus (WSMV)-infected and non-infected transgenic and non-transformed wheat [ Triticum aestivum L. (Poaceae)]. Although R. padi is not a WSMV vector, it is known to colonize WSMV-infected wheat plants. Two transgenic soft white winter wheat genotypes, 366-D03 and 366-D8, that express the WSMV coat protein gene, and the WSMV-susceptible non-transformed cultivar Daws were tested. All genotypes showed disease symptoms when infected with WSMV. Whereas plant height was significantly reduced on virus-infected compared to non-infected plants of all genotypes, virus-infected transgenic plants exhibited lower virus titer and lower disease rating scores than Daws. No significant effects of WSMV infection or genotypes were observed on the length of R. padi nymphal development period, nor on their pre-, and post-reproductive periods. Rhopalosiphum padi reproductive period was significantly longer on Daws infected with WSMV than on non-infected plants of this cultivar. In contrast, there were no significant differences in length of R. padi reproductive period between virus-infected and non-infected transgenic plants within a genotype. Rhopalosiphum padi daily fecundity was significantly lower and adult longevity significantly longer on virus-infected than on non-infected plants of all genotypes. Total aphid fecundity and intrinsic rate of increase were not significantly different among treatments. The percentage of winged aphids that developed was greater on WSMV-infected compared to non-infected plants within a genotype. Results indicate that both virus infection status of plants and wheat genotype influence the life history of R. padi.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

Rapid assessment of Autographa californica nuclear polyhedrosis virus infection of Sf-9 insect cells by analyzing suppression of reagent-induced apoptosis

Spodoptera frugiperda Sf-9 insect cells that undergo apoptosis by the treatment of apoptosis-inducing reagents were individually determined as `comet cells' having a tail of fragmented DNA during single cell gel electrophoresis, the fragmented DNA being migrated from the cells under an electric field of the electrophoresis. However, the apoptosis induction of the cells infected with a recombinant strain of Autographa californica nuclear polyhedrosis virus (AcMNPV) was blocked, probably by an intrinsic anti-apoptotic p35 gene of the virus, because the virus-infected cells did not have a tail of fragmented DNA on a single cell gel electrophoresis. The virus-infected cells were individually discriminated from non-infected cells by determining the anti-apoptotic nature of the cells. At higher multiplicity of infection and under better aeration conditions of virus-infected cultures, the apoptosis-suppressive ratio, which represented a ratio of non-comet cells, was increased more rapidly. This apoptosis-suppressive behavior was a good benchmark for assessing successful infection of insect cells with AcMNPV during very early infectious period and forecasting the subsequent production of recombinant proteins.