The drosophila X virus contains a 1-microM double-stranded RNA circularized by a 67-kd terminal protein: high-resolution denaturation mapping of its genome

The effect of urea on Drosophila X virus (DXV) is presented and analyzed by electron microscopy. At 4 M urea a RNA-protein complex is liberated consisting of one segment of double-stranded (ds) RNA which is maintained in a circular form inside the virion by a protein of 67 kd. The RNA-protein complex was purified by chromatography and had a tendency to form very spectacular flower-like structures. By crosslinking the RNA inside the virus with psoralen and uv radiation it is shown that the RNA was double-stranded in situ. A new method for partial denaturation mapping of double-stranded nucleic acids by electron microscopy is described and was applied to DXV RNA. Under these conditions it is shown that native and denatured regions as small as 30 RNA base pairs (bp) can be visualized. In the case of DXV RNA it was observed that the RNA possessed at one extremity a GC-rich region of 30 by followed by an AU-rich region of 160 bp. These molecules were observed in a conventional transmission electron microscope and a scanning transmission electron microscope under dark-field illumination. Absolute electron microscope length measurements and molecular weight determinations by gel migration revealed that the DXV RNA is one segment of ds-RNA of 0.97 μm in the electron microscopic conditions used in this work, giving a molecular weight of 2.2 × 10⁶ d which corresponds to 3170 RNA bp. An average melting temperature of 83.3° was obtained in 0.1 SSC. Viral protein analysis was performed on the virus and on the purified protein-RNA complex. The virus is made of six major proteins and two minor proteins which were revealed after in vitro iodination.     

Association of cowpea mosaic virus-induced double-stranded RNA with a cytopathological structure in infected cells

A cytopathological structure in cowpea mosaic virus (CPMV) infected cowpea leaves 4 days after inoculation was characterized ultrastructurally.The fraction of a homogenate of infected leaves sedimenting at 1000 g was fractionated on a discontinuous sucrose gradient consisting of layers of 20, 45, and 60% sucrose. The chloroplast fraction, which was found at the interface of the 20% and 45% sucrose layers, known to contain 70–90% of all CPMV-RNA hybridizable material, was further fractionated on a discontinuous gradient consisting of layers of 37, 39, 41, 43, and 45% sucrose. The material at the interface of the 37% and 39% sucrose layers (Fraction I) and that at the interface of the 39% and 41% layers (fraction II) contained 90% of all structures resembling the cytopathological structures and less than 30% of all chloroplasts. Fractions I and II contained together approximately 90% of all CPMV-RNA hybridizable material. The fractions collected from the 43% and 45% sucrose layers and the pellet (fraction III) contained more than 70% of the chloroplasts, less than 10% of the cytopathological structures, and less than 10% of the CPMV-RNA hybridizable material. It was concluded that the CPMV-dsRNA is associated with the cytopathological structures in the cytoplasm rather than the chloroplasts.Autoradiography performed on sections of intact tissue and on sections of pellets of fractions I, II, and III, from tissue treated with ³H-uridine, thymidine, and actinomycin D, presented strong evidence that CPMV-RNA replication is associated with the vesicular constituent of the cytopathological structures.     

Rous sarcoma virus p19 binds to specific double-stranded regions of viral RNA: effect of p19 on cleavage of viral RNA by RNase III.

The extent of binding of purified RSV(Pr-C) p19 and p12 to a variety of RNAs was measured using a sensitive nitrocellulose filter binding assay which is capable of detecting binding reactions with association constants as low as 3 × 10⁶ liters × mol^?1 (Hizi, A., Leis, J. P., and Joklik, W. K. 1977). RSV p19 bound 60 and 34 S RSV (Pr-C) RNA with association constants of 5.1 × 10¹¹ and 1.8 × 10¹⁰ liters × mol^?1. RSV p19 bound preferentially to specific double-stranded regions of the RNA since: (a) The association constant for Neurospora nuclease-digested 34 S RNA was the same as for untreated RNA; (b) the association constant for 34 S RNA partially digested with Escherichia coli RNase III (which is specific for double-stranded RNA regions) was 30-fold lower than for untreated RNA; (c) p19 prevented cleavage of 34 S RSV-RNA by E. coli RNase III; (d) p19 bound cell precursor RNAs containing RNase III-sensitive sites, but not mature RNAs lacking RNase III-sensitive sites. On the other hand, purified RSV p12 bound all RNAs tested with association constants roughly proportional to their molecular weights. A possible function for p19 in regulating the processing of viral RNA and its subsequent translation has been proposed.     

Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: selective removal of host RNA polymerase from membranes containing CCMV RNA replicase.

Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg²⁺ ions for activity. Manganese ion was a poor substitute for Mg²⁺. Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg²⁺ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.     

A possible replicative form of brome mosaic virus RNA 4

A double-stranded RNA of a size expected for the replicative form of BMV RNA 4, the smallest of the four RNAs of brome mosaic virus, was isolated from infected leaves by a procedure involving removal of single-stranded RNA by precipitation in 2 M LiCl, followed by cellulose chromatography and polyacrylamide gel electrophoresis. Like the other double-stranded RNAs of BMV, it had characteristics of a replicative form, such as distinctive elution patterns from cellulose and hydroxyapatite columns, and purple staining with toluidine blue O. It coelectrophoresed in 2.5% polyacrylamide gels with double-stranded RNA synthesized in vitro by Qβ replicase with BMV RNA 4 as template. The origin and mode of replication of BMV RNA 4 is discussed.     

Novel circular forms of mengovirus-specific double-stranded RNA detected by electron microscopy

Examination of mengovirus replicative form (RF) RNA by electron microscopy has revealed a substantial frequency of circular double-stranded RNAs which have an unusual projection on their circular contour. Circular structures comprised 14–16% of the genome-length double-stranded RNA in different preparations and have a contour length of 2.25 ± 0.08 μm corresponding to 8840 ± 310 nucleotide pairs in the A-form nucleic acid. The projection from the circular contour was typically found on more than 98% of the molecules and has an average heterogeneous length of 0.08 ± 0.02 μm (300 ± 80 nucleotide pairs). Linear duplex molecules with opposed ends were also detected at frequencies of 5–9% in different preparations and appear to be derived from dissociation of circular structures at the site of the projection. These linear duplexes had a length 2.32 ± 0.08 μm (9100 ± 310 nucleotide pairs) which is slightly greater than the contour length of circular structures excluding their projection. Linear dimers (3–6% of total), circular dimers (0.3%), and catenanes of two and three circular RF molecules, respectively, have been detected at low frequencies; such molecules usually contained a projection. The integrity of circular structures was destroyed by mild treatment with ribonucleases A, T1, or T2 or by incubation in 50% formamide or 40% formamide plus 2 M urea but was unaltered by treatment with DNase I or proteinase K. When examined by electron microscopy in the presence of formamide or formamide plus urea, approximately 14–17% of the linear duplex molecules were detected which contain one single-strand terminus typically less than 500 nucleotides in length. We suggest that circular structures are formed by noncovalent association of one single-strand terminus and one double-strand terminus of linear duplex molecules and that this association produces circular structures with the projection described at the site of association. A fixed fraction of linear RF RNA molecules in any given preparation contains termini suitable for association into circular structures that may have biological significance in the replication of picornaviruses.     

A reexamination of influenza single-and double-stranded RNAs by gel electrophoresis.

Influenza virus single- and double-stranded RNAs have been examined by polyacrylamide-gel electrophoresis on slab gels. In both cases eight RNA segments have been demonstrated, and these are grouped as three large, three intermediate, and two small segments. The single-stranded RNAs were electrophoresed in gels containing 6 M urea, and the molecular weight of the entire single-stranded RNA genome of influenza virus was estimated to be 5.9 × 10⁶.     

Cherry leafroll virus infections are affected by a satellite RNA that the virus does not support

Previous research showed that tobacco ringspot virus (TobRV), a member of the nepovirus group, acts as a supporting virus for the 359-nucleotide residue satellite tobacco ringspot virus RNA (STobRV RNA), resulting in STobRV RNA replication and its encapsidation in TobRV coat protein. In some hosts STobRV RNA decreases the yield of TobRV and the severity of TobRV-induced symptoms. We report here that inclusion of STobRV RNA in an inoculum of cherry leafroll virus (CLRV), another nepovirus, prevented the accumulation of CLRV in the inoculated leaves of cowpea (Vigna unguiculata) and caused the symptoms to be less severe than those induced when CLRV was inoculated alone. CLRV spread to, and increased in, uninoculated, developing leaves whether or not it was coinoculated with STobRV RNA. STobRV RNA was not detected in CLRV particles or in extracts of infected tissue from the coinoculated plants, indicating that CLRV does not support STobRV RNA. STobRV RNA strongly interfered with the in vitro translation of the RNAs of CLRV and of cowpea mosaic virus (CPMV). Coinoculation of STobRV RNA and CPMV had no detected effect on infections by CPMV, so inhibition of translation in vitro and of replication in vivo were correlated only for CLRV. Results from a new in vitro assay for proteolytic processing of CLRV polyproteins gave no indication of an effect of STobRV RNA on this reaction.     

Participation of the Cowpea mosaic virus protease in eliciting extreme resistance

Extreme resistance of Arlington line cowpea (Vigna unguiculata) to Cowpea mosaic virus (CPMV) is under control of a dominant locus designated Cpa. We transiently expressed, using Tomato bushy stunt virus (TBSV) vectors and Agrobacterium tumefaciens, in nearly isogenic Cpa/Cpa and cpa/cpa cowpea lines, sequences from RNA1, the larger of two CPMV genomic RNAs. Activation of a Cpa-specific response mapped to the CPMV 24K protease (24KPro). Mutational analysis of the 24KPro gene implicated protease activity, rather than 24KPro structure, in Cpa-mediated recognition of CPMV invasion. A 24KPro with alanine replacing the active site cysteine [24KPro(C-A)], but not wildtype 24KPro, accumulated after agroinfiltration of the corresponding binary vector constructions into Cpa/Cpa cowpea. In cpa/cpa cowpea, both protease versions accumulated, with 24KPro(C-A) in greater abundance. Thus, enzymically active 24KPro was recognized by both cowpea genotypes, but in Cpa/Cpa cowpea the suppression of 24KPro accumulation was very strong, consistent with extreme resistance to CPMV.     

The cowpea mosaic virus RNA replication complex and the host-encoded RNA-dependent RNA polymerase-template complex are functionally different

Purification of the putative cowpea mosaic virus (CPMV) RNA replicase previously started with an RNA-dependent RNA polymerase activity which had been solubilized from a crude membrane fraction of CPMV-infected cowpea leaves by extraction with a Mg2+-deficient buffer. This led to the identification of a host-encoded, 130,000-dalton monomeric enzyme, the activity of which was highly enhanced upon infection. As the role of this enzyme in viral replication was questionable, we reverted to the template-associated RNA-dependent RNA polymerase in the crude membrane fraction in order to characterize in detail its in vitro products. We now demonstrate that the crude membrane fraction of CPMV-infected cowpea leaves harbors two functionally different, RNA-dependent RNA polymerase activities that are both associated with endogenous template RNA and can be separated from each other without affecting their distinct properties. One of the RNA polymerase activities was specific for CPMV-infected leaves and constituted a CPMV RNA replication complex; enzyme activity in vitro allowed for the completion of nascent chains initiated in vivo. Full-length viral RNAS (B- and M-RNA) were produced which were recovered mainly in double-stranded form. Solution- and Northern blot hybridization demonstrated that the in vitro-labeled RNA chains were viral RNAs of positive polarity. The other template-associated RNA-dependent RNA polymerase activity occurred in both uninfected and infected leaves and transcribed in vitro endogenous plant and viral RNAs only into small RNAs (4-5 S) of negative polarity. Northern blot analysis revealed the RNA products of plant origin to be transcribed from two major RNA templates of approximately 0.26 and 0.14 X 106 daltons, respectively. Washing of the crude membrane fraction in a Mg2+-deficient buffer did accomplish the complete release of the low-molecular-weight RNA-synthesizing activity but did not solubilize the CPMV RNA replication complex. We tentatively conclude that the RNA-dependent RNA polymerase which has previously been purified from the buffer-soluble fraction and has been identified as a host-encoded enzyme is not involved in viral RNA replication. Solubilization of the viral replication complex was achieved with Triton X-100. By taking advantage of the characteristic conformation of the replication complex, we applied Sepharose 2B chromatography as a highly efficient and simple means for purifying the detergent-solubilized complex. We anticipate that this purification step should also be applicable to replication complexes of other RNA viruses.     

Structural studies of the RNA component of the poliovirus replication complex. II. Characterization by electron microscopy and autoradiography.

The endogenous RNA component of the purified poliovirus replication complex was characterized in the electron microscope after cytochrome spreading. This RNA species has a double-stranded RNA core equal in length to poliovirus replicative form RNA, with 0 to 6 single-stranded tails per molecule. DNase I, RNase, and base treatment confirmed the double-stranded RNA nature of these molecules, which are not observed in extracts from uninfected or infected, guanidine-inhibited cells. Electron microscope autoradiography verified that these double-stranded RNA structures are the site of in vitro and in vivo viral RNA synthesis. After RNA synthesis in vitro, the double-stranded core is unchanged, but the number of tails decreases and the branch points are localized towards the ends of molecules. These results demonstrate that the RNA component of the active purified replication complex is analogous to replicative intermediate RNA and favor the double-stranded model for the in vivo poliovirus replication complex.     

Termperature-sensitive mutants of pseudorabies virus with differential effects on viral and host DNA synthesis

Mouse kidney cells infected with polyoma virus were labeled for 20 min with [5,6-³H]uridine late in infection. The rapidly-labeled RNA was extracted from whole cells and from the Hirt SDS/high salt supernatant fraction. The RNA was self-annealed and became resistant to ribonuclease digestion. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded RNA by several different criteria: resistance to RNase and to the combined effects of RNase and DNase, the characteristic buoyant density of double-stranded RNA in Cs₂SO₄, a buoyant density increase upon denaturation to that of single-stranded viral RNA marker, an inability to hybridize with complementary DNA unless denatured, and a high T_m value with a sharp transition to RNase sensitivity. Separated strands of the double-stranded RNA hybridized with high efficiency with component I of polyoma DNA. After self-annealing in formamide at low temperature, from 18 to 30% of the total rapidly-labeled viral RNA of infected cells sedimented at 11 S; 11 S corresponds in size to about 30% of the polyoma DNA. The extraction procedure for double-stranded viral RNA also yields double-stranded cellular RNA, but the double-stranded viral RNA can be further purified by using the Hirt supernatant, the RNaseresistance of the viral RNA coupled with its higher T_m, its greater sedimentation coefficient.These observations indicate that, late in infection of mouse cells, polyoma DNA is transcribed symmetrically over a considerable portion of its length, yielding self-complementary RNA that is distinguishable from the cellular self-complementary RNA.     

Inverted complementary terminal sequences in single-stranded RNAs and snap-back RNAs from vesicular stomatitis defective interfering particles.

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.     

Strand assignment of polyadenylated nuclear RNAs synthesized early in infection with adenovirus 2

Regions of the adenovirus 2 genome coding for early cytoplasmic RNAs also specify polyadenylated nuclear RNAs larger than the cytoplasmic RNAs [Craig, E. A., and Raskas, H. J. (1976). Cell 8, 205–213]. These large polyadenylated nuclear RNAs were analyzed further by hybridization studies with unique viral DNA fragments generated by digestion with endo R·Eco R1, endo R·HindIII, and endo R·Sma I. Hybridization-inhibition experiments were performed with ³H-labeled nuclear RNA fractionated by size. The results demonstrated that the larger nuclear RNAs are derived from the same DNA strand as the cytoplasmic RNAs and that they contain most if not all the sequences present in cytoplasmic RNAs. Hybridization of the larger polyadenylated nuclear RNAs transcribed from the l strand of Eco R1-C and the r strand of Eco Rl-D was inhibited approximately 80% by cytoplasmic RNA; hybridization of RNA from the l strand of Eco R1-B was inhibited only 50% by cytoplasmic RNA. From the molecular weights of these nuclear RNAs and the results of the hybridization-inhibition studies, it appears that these nuclear RNAs contain from 600 to 2300 nucleotides which are not detected in cytoplasmic RNA.     

Association of tobacco etch virus related RNA with chloroplasts in extracts of infected plants

The RNA in various subcellular fractions of tobacco etch virus (TEV) infected tissue was analyzed for the presence of complementary viral RNA, and double-stranded viral RNA by hybridization with 32P-labeled viral RNA or cDNA probes. Although viral RNA was detected in several cellular fractions, the complementary RNA of full-length size was found exclusively associated with fractions containing chloroplasts. Treatment of RNAs with RNase before hybridization suggested that the virus-related complementary RNA was present in double-stranded form.     

Analysis of RNA associated with the poliovirus RNA replication complexes

Two size-classes of RNA replication complexes were isolated from the smooth microsomal fraction of poliovirus-infected HeLa cells: a complex that sediments at less than 70 S and another in the region from 100 S to 300 S. The virus-specific RNAs associated with the replication complexes were characterized by velocity sedimentation, acrylamide-agarose gel electrophoresis, and hybridization with poliovirus RNA. In vivo, the large replication complex contains predominantly single-stranded 35 S RNA, but only 8% of the RNA anneals to viral RNA. The small replication complex contains predominantly double-stranded RNA, and over 60% of this RNA anneals to viral RNA. These results suggest that the small replication complex may be the primary site of complementary RNA synthesis in the cell.About 50% of the RNA synthesized in vitro by the large replication complex is single-stranded RNA, whereas almost all of the RNA synthesized in vitro by the small replication complex is double-stranded RNA. However, between 20 and 30% of the RNA synthesized in vitro by both the large and the small replication complexes is complementary RNA. Possible reasons for the differences between the in, vivo and in vitro function of the replication complexes are discussed.     

Initiation factor preparations from poliovirus-infected cells restrict translation in reticulocyte lysates

The translation of poliovirus RNA and vesicular stomatitis virus (VSV) mRNAs was studied in reticulocyte lysates supplemented with uninfected HeLa cell or polio-infected HeLa cell ribosomal salt wash as a source of initiation factors. Ribosomal salt wash from uninfected HeLa cells supported translation of both RNAs. Similar preparations from polio-infected HeLa cells stimulated translation of polio RNA; however, the translation of VSV mRNAs was completely restricted. Lysates programmed simultaneously with polio RNA and VSV mRNA translated both RNAs, although VSV translation was predominant. Addition of polio-infected cell ribosomal salt wash resulted in translation of polio RNA exclusively. The infected cell salt wash did not prevent formation of 40 S·Met-tRNA_f^Met complexes, but did prevent binding of labeled VSV mRNA to 40 S initiation complexes. The ability to restrict VSV mRNA translation resided in the 40% ammonium sulfate fractional precipitate prepared from infected cell salt wash. Similar fractionation of ribosomal salt wash mixed with labeled poliovirus double-stranded RNA indicated that this component was also found in the 40% ammonium sulfate fraction. The effects on translation of double-stranded RNA and the restrictive activity of infected cell salt wash were compared: mRNA specificity, kinetics of inhibition, nuclease sensitivity, and relief of inhibition by excess double-stranded RNA differed. From these comparisons, the possibility that double-stranded RNA caused the restriction of VSV mRNA translation was excluded.