Bunyamwera virus II. The generation and nature of defective interfering particles

Autointerference with the replication of Bunyamwera virus was produced by propagation of virus in BHK-21 cells. To investigate this phenomenon, standard and defective stocks of Bunyamwera virus were generated. The defective viral stock only inhibited the replication of homologous standard virus and this interference was host cell-dependent. Interference was high in BHK and Vero cells but greatly reduced in MDBK and Aedes albopictus cells.Evidence was obtained suggesting that Bunyamwera defective particles exerted an inhibitory effect at the level of viral genomic replication. Infection of BHK cells with a mixture of standard and defective Bunyamwera virus resulted in an alteration of the intracellular profile of viral RNA. Incorporation of radiolabeled precursors into the higher molecular weight viral RNAs (33 and 26 S) was greatly reduced, whereas labeling of the virus-specific 16 S species was unaffected.Virus particles produced under varying degrees of autointerference were studied by isopycnic centrifugation. As the degree of autointerference was increased, the viral particles became more heterogenous with respect to their RNA content. Under conditions of multiple viral passages at high multiplicity in BHK cells, two major classes of virus particles were produced, one broad band at ? = 1.19 g/cm³ and another at 1.17 g/cm³ Particles at 1.17 g/cm³ were noninfectious, contained only 16S viral RNA, and interfered with the replication of standard (1.19 g/cm³) Bunyamwera virus.     

Physico-chemical characterisation of rinderpest virus

Rinderpest virus grown in Vero cells was purified by equilibrium sedimentation in potassium tartrate gradients. The characteristics of the virus and its RNA dependend on the passage history of the virus. Undiluted passage virus had a density ofc 1.21 g/ml in potassium tartrate and contained RNA sedimenting atc. 20S and 5S. Virus obtained by dilute passage had a density ofc. 1.24 g/ml and contained 50S in addition to 20S and 5S RNA. Both diluted passage and undiluted passage viruses contained polypeptides with molecular weights of 98, 79, 75, 66, 48, 43 and 37×10³. Trace amounts of polypeptides with molecular weights of 29 and 20×10³ were also detected. The molecular weights of the major polypeptides are similar to those of the polypeptides present in measles and canine distemper viruses.     

Fractionation of the double-stranded RNA of maize rough dwarf virus subviral particles

Double-phase phenol-SDS, single-phase phenol-SDS and sodium perchlorate methods were employed to isolate the RNA from purified maize rough dwarf virus subviral particles (MRDV-SVPs). The three different procedures gave identical results. MRDV-RNA sedimented as two peaks during sucrose density gradient centrifugation. Fractionation of the RNA by polyacrylamide gel electrophoresis resulted in 9 distinct classes, all double-stranded. Their molecular weights, expressed in daltons (× 10⁶) were 1.11, 1.19, 1.21, 1.42, 1.73, 2.06, 2.24, 2.33, 2.58.     

Characterization of hepatitis B virus (Dane particle)

Hepatitis B virions (Dane particles) were purified from the sera of chronic HBsAg carriers by consecutive rate-zonal and isopycnic centrifugations in sucrose gradients using HBsAg, HBcAg and endogenous DNA polymerase activities as specific markers. Purified Dane particles, radiolabelled with Na ¹²⁵I by the chloramine-T procedure, had a higher buoyant density in CsCl (1.28 g/cm³) than unlabelled particles (1.26 g/cm³) and an estimated sedimentation coefficient of 280 s. ¹²⁵I-Dane particles were fully precipitated by anti-HBs and not by anti-HBc sera. Heavy and light density core particles were purified from heavy and light density populations of Dane particles and radioiodinated. The iodinated polypeptides of Dane particles and HBcAg were compared with those of the iodinated 22-nm form of HBsAg by SDS-PAGE. Iodinated Dane particles contained seven polypeptides with molecular weights of 18,000, 23,000, 26,000, 34,000, 43,000, 48,000 and 115,000. Heavy and light core particles contained three polypeptides with molecular weights of 18,000, 25,000 and 37,000.     

dsRNA associated with virus-like particles in Eimeria spp. of the domestic fowl

 RNA segments, identified as double-stranded, were found in sporozoites of the Guelph strains of Eimeria acervulina, E. brunetti, E. maxima and E. necatrix and in 8 of 11 strains of E. acervulina obtained from poultry houses across the United States. These RNAs were resistant to RNase A digestion in the presence of high salt concentrations (0.3 M NaCl). On agarose-gel electrophoresis, E. acervulina had one obvious band at 1.7 kb and a faint band at 3.5 kb; E. brunetti had two bands at 2.1 and 3.3 kb, respectively; E. maxima had one band at 4.5 kb; and E. necatrix had two major bands at 4.5 and 5.6 kb, respectively. No dsRNA band was seen in the three strains of E. tenella examined. Virus-like particles were purified by cesium chloride density centrifugation of homogenates of E. necatrix sporulated oocysts. The fraction at peak virus concentration had a buoyant density of 1.39 g ml^–1. These virus-like particles were icosahedral, had no envelope and measured 42–44 nm in diameter. Only one RNA band at 5.6 kb was observed when nucleic acids from gradient fractions containing virus were subjected to electrophoresis. The 4.5-kb dsRNA segment of E. necatrix was not associated with a virus-like particle. Received: 3 November 1995 / Accepted: 10 January 1996     

Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59)

We have determined the kinetics of virus production and virus-specific RNA synthesis in Sac(?) cells infected with mouse hepatitis virus strain A59 (MHV-A59). Immunofluorescence showed that all cells became infected at a multiplicity of 10 PFU/cell. The virus was concentrated and purified to obtain the high titered stocks needed for these one-step growth experiments. Release of virus into the culture medium started 4 hr after infection (pi) and was complete at 10 hr pi. Synthesis of virus-specific RNA, measured by the incorporation of [³H]uridine in the presence of 1 μg/ml actinomycin D, also started at 4 hr pi and its maximum rate occurred between 6 and 8 hr pi. RNA labeled during this period was isolated from infected cells. About 50% of this RNA bound to oligo(dT)-cellulose; this material was denatured with glyoxal-dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. Seven RNA species with the following molecular weights were present: 5.6 × 10⁶ (RNA1), 4.0 × 10⁶ (RNA2), 3.0 × 10⁶ (RNA3), 1.4 × 10⁶ (RNA4), 1.2 × 10⁶ (RNA5), 0.9 × 10⁶ (RNA6), and 0.6 × 10⁶ (RNA7). RNA1 comigrated with the viral genome. Artifacts caused by defective interfering particles or breakdown of RNA were excluded. To determine whether these RNA species were functional as messengers in infected cells, virus-specific RNAs present in polyribosomes were analyzed. EDTA treatment was used to discriminate between RNA present in polyribosomes and in EDTA-resistant, presumably ribonucleoprotein, particles. Most (91%) of RNA1 was present in EDTA-resistant particles; the remainder and all other RNAs synthesized between 6 and 8 hr pi were present in polyribosomes. We conclude that MHV-A59 has six subgenomic mRNAs. Since the total molecular mass (11.1 × 10⁶ daltons) of these messengers is about twice that of the viral genome, sequence homologies must exist between the mRNAs. The position of these homologous regions and the translation products of each of the mRNAs remain to be determined.     

Prunus necrotic ringspot virus as a multicomponent system.

When centrifuged in sucrose density gradients, Prunus necrotic ringspot virus separated into three zones of nucleoprotein particles with sedimentation rates in 0.03 M Tris, pH 7.5, of 72, 90, and 95 S. Bottom particles were slightly infectious; middle and top particles were noninfectious. Mixing of bottom and middle particles markedly increased infectivity, but additions of top particles decreased infectivity. A host range determinant conditioning infection of Vigna sinensis was carried in bottom particles. In 8 M urea, four bands of RNA were resolved by polyacrylamide-gel electrophoresis. After formaldehyde modification, three bands were resolved with molecular weights estimated as 0.95 × 10⁶, 0.66 × 10⁶, and 0.31 × 10⁶.     

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat

Summary.  Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 × 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 × 10⁶). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 ×  10⁶. The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5′ co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear. Received February 22, 2000 Accepted June 23, 2000     

Isolation and characterization of entomopox virions from virus-containing inclusions of Amsacta moorei (Lepidoptera: Arctiidae)

Preparations of Amsacta moorei entomopox virions were obtained from virus-containing inclusions (VCI) by using a carbonate-thioglycolate solution (pH 10.7–11.5). The virions possessed a uniform coat (“halo”) surrounding the viral envelope and exhibited an RNA polymerase activity. The “halo” could be removed by prolonged exposure to the carbonate-thioglycolate solution. Virions obtained by this treatment, however, possessed low infectivity and no detectable RNA polymerase activity. Removal of the “halo” by trypsin resulted in virions which possessed RNA polymerase activity and relatively high infectivity.Preparations of particles with and without the “halo” were similar in percent DNA, protein per OD₂₆₀, number of particles per OD₂₆₀, and RNA polymerase activity. Particles without the “halo,” however, were less dense (1.262 g/cm³) in CsCl than those with the “halo” (1.282 g/cm³) and 15–45 times more infective.Parallel studies of “nonhaloed” Amsacta virions (trypsin-treated) and vaccinia virions showed that both viruses contained similar amounts of protein per OD₂₆₀, but Amsacta virions contained only 36% of the DNA found in vaccinia.     

The purification of rubella virus (RV) and determination of its polypeptide composition

Rubella virus (RV) has been propagated in murine fibroblasts (L cells) and purified using two Renografin gradients. The virus was grown in the presence of 2 μCi/ml [³H]uridine, pelleted from tissue culture media 6 days postinfection, and applied to a 25–45% discontinuous gradient. A single, sharp band was observed at the interface. This band was collected and applied to a 30–45% continuous gradient which separated intact labeled virions from ³H-labeled, light density material. Infectivity was measured using a modified hemadsorption assay. A recovery of 90% of the RV infectivity was achieved by these methods. Purified virions obtained in this way were dissociated, labeled with ¹²⁵I, immune precipitated with rubella-specific antiserum, and subjected to polyacrylamide slab gel electrophoresis and autoradiography. Four polypeptides were observed with molecular weights of 44, 41, 24, and 19 × 10³ designated as VP44, VP41, VP24, and VP19, respectively.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. I. Physical and chemical properties

Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 10⁶,1.10 × 10⁶, 0.90 × 10⁶, and 0.43 × 10⁶ daltons, and CMV-RNA, species of 1.26 × 10⁶, 1.10 × 10⁶, 0.77 × 10⁶, and 0.34 × 10⁶ daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.     

Adeno-associated virus autointerference.

We have analyzed an autointerference phenomenon exhibited by adeno-associated virus type 2 (AAV) when grown in KB cells coinfected with adenovirus type 2 as the helper. Infectious AAV particles that banded at 1.41 g/cm³ in CsCl were purified by three cycles of centrifuging in CsCl equilibrium gradients. When cells were infected with an increasing multiplicity of these AAV particles there was a corresponding decrease in production of infectious progeny AAV. There was also an AAV multiplicity-dependent inhibition of production of infectious adenovirus and inhibition of Ad DNA replication. The viral DNA in the Hirt supernatant fraction extracted from cells infected with different multiplicities of AAV was analyzed in neutral sucrose gradients. At low multiplicities of infection with AAV, the main AAV DNA species synthesized was the mature 14.5 S (standard) viral genome. In higher multiplicity infections with AAV increasing amounts of aberrant 10 S AAV DNA molecules accumulated and the proportion of 14.5 S AAV DNA decreased. Restriction endonuclease cleavage showed that the 10 S DNA was enriched for the left- or right-hand terminal regions of the AAV genome. These molecules may be analogous to the previously characterized aberrant DNA molecules found in light-density AAV particles. Thus, the AAV autointerference is correlated with production of the aberrant deleted AAV genomes.     

Biochemical and genetic characteristics of germiston virus

Germiston virus grown in BHK 21 cells has an estimated particle density of 1.19 glcm³ in sucrose gradient. By polyacrylamide gel electrophoresis, viral RNA profile shows three single-stranded RNA segments with molecular weights of 3.8 × 105, 2.5 × 105, and 0.5 × 105. This virus possesses three major proteins with molecular weights of 125 x 103, 27 × 103, and 18 × 103 and a minor one of 185 x 103. Eight temperature-sensitive (ts) mutants isolated by chemical mutagenesis are distributed in two complementation-recombination groups with high frequencies of recombination.     

Purification and some physicochemical properties of sonchus yellow net virus

Sonchus yellow net virus (SYNV) was purified from a Nicotiana hybrid by Celite filtration and sucrose density gradient centrifugation. Infectious preparations sedimented at 1044 S in linear-log gradients and banded at 1.183 g/ml in sucrose equilibrium gradients. Electron microscopy of purified preparations revealed bacilliform particles (94 × 248 nm). The virions had internal cross striations with a periodicity of about 4.1 nm and surface projections about 6 nm long. The molecular weight of the virion, estimated from size and density, was about 9 × 10⁸. Nucleic acid from sodium dodecyl sulfate-disrupted virions was susceptible to RNase, sedimented in sucrose gradients at 44 S, and had a molecular weight of 4.42 × 10⁶ as estimated by polyacrylamide-gel electrophoresis. Four major polypeptides with average molecular weights of 76,800, 63,800, 45,500, and 39,500 were detected by gel electrophoresis. SYNV preparations reacted in gel diffusion tests with a homologous antiserum but not with antisera to broccoli necrotic yellows virus, lettuce nectrotic yellows virus, or sow thistle yellow vein virus.     

Molecular weight estimates of vesicular stomatitis virus ribonucleic acids from virions,defective particles,and infected cells

Summary Ribonucleic acid from vesicular stomatitis virus (VSV) virions (B particles), defective particles (T), and infected cells was examined by polyacrylamide gel electrophoresis, and apparent molecular weights were estimated. B particle RNA of 4.0×10⁶ daltons from Indiana strain L and from Cocal virus was distinguishable from RNA of 4.5×10⁶ daltons from Indiana strain BT-78 and several strains of New Jersey VSV. Abundant T particle RNA of 1.0×10⁶ daltons, was present in one Indiana L stock and was absent in a plaque purified stock. Cocal virus had T particle RNA of 0.7×10⁶ daltons, and one stock of Ogden strain New Jersey VSV had T particle RNA of 1.2×10⁶ daltons. Little or no T particle RNA was detectable in several other VSV stocks. Major RNA components with apparent molecular weights, 2.4, 0.66, and 0.31×10⁶ daltons in cells infected with Indiana strain L were complementary to virion RNA. The size relationships of B particle RNA from Indiana L and New Jersey strains were examined by sucrose density gradient sedimentation of undenatured and of formaldehyde denatured RNA. Sedimentation of undenatured RNA led to a conflict with gel electrophoresis results, inferring New Jersey had a lower molecular weight than Indiana L, but upon denaturation the relation was consistent with electrophoresis results.     

Unusual attachment behavior exhibited by a urea-resistant mutant of poliovirus type 1.

A urea-resistant mutant of type 1, LSc poliovirus, called LSc-U^r, always produced about 30% of the progeny and 50% of the viral-specific RNA in infected cells as compared to its parent virus. LSc-U^r virus was found to attach normally at low multiplicities of infection (m.o.i.) but exhibited autointerference at higher m.o.i. For example, at an m.o.i. of 10, less than 25% of the added virions was cell associated after 90 min. Neither parental LSc nor Mahoney poliovirus exhibited unusual attachment kinetics under comparable conditions. In addition, LSc-U^r (m.o.i. = 10) interfered markedly with the attachment of Mahoney poliovirus. These findings were interpreted as resulting from a high particle/PFU ratio for the LSc-U^r virus. LSc-U^r virus partially purified by differential and sucrose gradient centrifugations still exhibited attachment autointerference at high m.o.i. in contrast to virus purified by CsCl banding which showed normal attachment and a much reduced ability to interfere with the attachment of other polioviruses. These results provide circumstantial evidence that LSc-U^r virus preparations contain virion-like particles (s_20,w of 120–150 S) which compete with the attachment of virions to cell receptors and which are degraded by high concentrations of CsCl.     

Purification and partial characterization of sugar beet yellows virus

A technique was devised for obtaining purified infectious preparations of sugar beet yellows virus (SBYV) particles. As the use of mechanical blending resulted in shearing of particles, infected leaves were hand ground in a mortar; the extracts were clarified with bentonite, concentrated using polyethylene glycol, and further purified by rate zonal and isopycnic gradient centrifugation. The purified particles had a modal length of 1270 nm, sedimented at 110 S, had an unusual ultraviolet absorption spectrum and contained about 5% RNA. The molecular weight of the infectious RNA, estimated by sedimentation and by gel electrophoresis was about 4.3 × 10⁶; that of the coat protein subunit was about 23,500. These properties are discussed in relation to the structure of the virus particles and also are compared with the properties of viruses allied to SBYV.     

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.     

Precursor-product relationship between nonglycosylated polypeptides of A and B particles of mouse mammary tumor virus.

Polypeptides and antigens of various preparations of intracytoplasmic A particles were analyzed in detail in comparison with those of B particles of mouse mammary tumor virus (MTV). SDS-polyacrylamide-gel electrophoresis (PAGE) revealed that B particles consisted of three major nonglycosylated polypeptides (B-p25, B-P15, and B-P7; numerals indicate calculated molecular weights in 10³ daltons) and six glycopeptides. All A-particle polypeptides were nonglycosylated. The particles purified by a conventional method contained seven major bands in the 70,000- to 37,000-molecular weight region, but their relative amounts varied from preparation to preparation. In most preparations, one other major band, A-p7 (an A-particle polypeptide with a molecular weight of 7000), was also observed. Thus, the polypeptide composition of A particles was quite different from that of B particles, having in common only one major band, p7, and three other bands of variable amounts, p43, p37, and p13. Those A particles that had been incubated at 37° for 20 hr showed a systematic change in PAGE pattern; major bands disappeared except for A-p43, A-p37, and A-p7, while a strong new band, A-p25, appeared. Consequently, incubated A particles were now similar to B particles in their PAGE pattern except for the absence of p15 in the-A particles. In spite of such a profound change in components, no ultrastructural alteration was observed in the A particles after incubation. Polypeptide conversion induced by incubation was completely inhibited by diisopropylfluorophosphonate (DFP). A particles purified in the presence of phenylmethanesulfonyl fluoride (PMSF) consisted of a single major polypeptide, A-p70. Incubation of these A particles resulted in generation of A-p15 in addition to A-p25 and A-p7 at the sacrifice of A-p70, although the rate of polypeptide conversion was much retarded. Antigenic analysis of individual polypeptides eluted from polyacrylamide gels shows that (i) B-p25, B-p15, and B-p7 carried distinct antigens; (ii) B-p25 and B-p7 were antigenically identical with A-p25 and A-p7, respectively; (iii) A-p70 carried all three of these different antigenicities of B particles; and (iv) other major polypeptides of A particles also carried these antigens in ways characteristic to each antigen. This indicates that three major internal components of B particles are generated from a common precursor, A-p70, through enzymatic cleavage and, hence, that A particles are the real pronucleocapsids of B particles. Present observations are discussed in connection with the precursor-product relationships proposed in other RNA tumor virus systems.