Production and characterization of the cores of the "R" strain of mosquito iridescent virus.

Cores of “R” strain of mosquito iridescent virus (RMIV) were produced in vitro by reacting intact virus with chymotrypsin. Isolation of the cores from undegraded virus and outer capsid protein was accomplished by density gradient and differential centrifugations. Negatively stained core particles had a diameter of 176.1 ± 6.0 nm when examined in the electron microscope. The density of the particles as measured in cesium chloride gradients was 1.33, and the s_20,w was 3126 as measured in the analytical centrifuge. The molecular weight of the cores was calculated to be 1.84 × 10⁹ daltons. Protein, DNA, and lipid analysis of the cores accounted for all but 48.0% of the protein of intact virus particles. SDS-polyacrylamide gel electrophoresis of the cores compared with that of intact virus showed that only a 55,000 molecular-weight protein was absent in the former. The cores did not infect larvae or an Aedes aegypti cell line, and serological comparisons of intact virus and cores showed the two did not share common surface antigens.     

Some properties of the genome of murine cytomegalovirus (MCV)

The DNA of murine cytomegalovirus (MCV) was analyzed, in comparison with T4 and herpes simplex (HSV) DNAs, by velocity sedimentation in neutral and alkaline sucrose gradients; and by centrifugation to equilibrium in cesium chloride. The molecular weight of MCV-DNA was calculated to be 132 × 10⁶, relative to 110 × 10⁶ for T4 DNA, and 85 × 10⁶for HSV DNA. Like other herpes viruses, the MCV genome possessed alkali sensitive single strand regions. However it was possible to obtain some of the MCV DNA molecules free of alkali sensitive regions. Intact MCV DNA banded in cesium chloride as a homogenous collection of molecules of density corresponding to 59% G + C. However, quarter molecules or smaller fragments banded at two discrete densities, corresponding to 57.5 and 61.5% G + C.     

Physicochemical properties of two distinct types of virus-like particles from Helminthosporium victoriae

Two serologically distinct types of virus-like particles (VLPs) were isolated from Helminthosporium victoriae. The first, which was isolated from three normal and two diseased isolates, sedimented at a rate of 190 S when centrifuged in linear-log sucrose density gradients. The second, which was found only in the two diseased isolates, had a sedimentation coefficient of 145 S. No VLPs were detected in two other normal isolates of the fungus. The 190 and 145 S VLPs both were polyhedral and 35 to 40 nm in diameter and had typical nucleoprotein absorbancy spectra. The 190 S type of VLP was electrophoretically distinct from the 145 S VLP when electrophoresed on either polyacrylamide gels or cellulose acetate strips. Equilibrium density gradient centrifugation in cesium chloride revealed a single density component for the 190 S VLPs with a buoyant density of 1.4325 g/cm³ The 145 S VLPs could be resolved into two to four components with buoyant densities of 1.3813 to 1.4138 g/cm³. SDS polyacrylamide gel electrophoresis of the 190 S VLPs revealed two major proteins of molecular weight 83,000 and 88,000 present in equimolar amounts. The 145 S type of VLP had three major proteins with molecular weights of 92,000, 97,000, and 106,000; these were also present in equimolar amounts. Both the 145 and 190 S VLPs contained double-stranded RNA (dsRNA). The 190 S type of VLP had a single species of dsRNA with a molecular weight of 3.0 × 10⁶. There were four classes of dsRNA associated with the 145 S VLPs, and their molecular weights were 2.4, 2.2, 2.1, and 2.0 × 10⁶.     

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.     

Size distribution and In vitro translation of the RNAs isolated from turnip yellow mosaic virus nucleoproteins

Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology12, 521–539, 1960). When analysed by polyacrylamide-gel eletrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 × 10⁶ daltons) was present only in nucleoproteins B₁ and B₂. Nucleoproteins B₀, B₀₀, and B₀₀₀ contained RNAs ranging in size from approx 1.3 to 0.28 × 10⁶ daltons. The 0.28 × 10⁶-dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B₀₀₀ and B₀₀. Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 × 10⁶-dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated.     

Purification and properties of Herpesvirus saimiri DNA

³H-Thymidine-labeled Herpesvirus saimiri (HVS) was purified from supernatant and cells of infected owl monkey kidney monolayer cultures. Pronase/SDS-extracted HVS DNA was characterized in neutral sucrose gradients. Cocentrifugation of this DNA with ¹⁴C-labeled T4-phage DNA resulted in s⁰₂₀, w = 58 ± 1.5 S as the sedimentation constant, corresponding to a molecular weight of 91 ± 5 × 10⁶ daltons. Unsheared HVS DNA banded in cesium chloride at 1.709 g/ml, but it broke down during the different manipulations to at least two double-stranded DNA molecules of largely different base composition which shared no sequence homologies. One part (42% of the total viral genome) had a density of 1.729 g/ml, corresponding to 70% cytosine plus guanine content, the other one (representing 58% of the intact molecule) banded at 1.694 g/ml, corresponding to 35% cytosine plus guanine.     

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.     

Biophysical and biochemical characterization of virus-like particles containing a high molecular weight ds-RNA from Helminthosporium maydis.

Spherical virus-like particles (VLPs_ isolated from Helminthosporium maydis ATCC 32450 are 48 in diameter and have three serologically identical components which have the following properties: s_20,w = 152, 212, and 283; ? (in CsCl) = 1.298, 1.378, and 1.438 g cm^?3; percentage of RNA = 0, 17, and 32; and molecular weight = 14, 17, and 21 × 10⁶. The nucleic acid of the fastest sedimenting component is double-stranded and has the following properties: s_20,w = 20.69; T_m = 79.6°; ? (in Cs₂SO₄) = 1.6062 g cm^?3.; molecular weight = 6.3 ± 0.5 × 10⁶.     

The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization of cytomegalovirions and dense bodies.

Nucleic acid isolated from subviral particles of Fiji disease virus (FDV) was identified as double-stranded (ds)-RNA by the following properties: (1) Positive orcinol reaction; (2) resistance to ribonuclease (RNase) in 1 × SSC (sodium chloride-sodium citrate buffer) but not in 0.1 × SSC; (3) susceptibility to RNase in 1 × SSC after thermal denaturation; (4) sharp thermal denaturation curve with a melting temperature of 76° in 0.01 × SSC; (5) buoyant density of 1.60 g/cm³ in Cs₂SO₄; and (6) no increase in ultraviolet absorption on treatment with formaldehyde at 37°. On electrophoresis in polyacrylamide gel, FDV-RNA separated into nine RNA segments with a total molecular weight of 15.3 × 10⁶.     

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.     

The generation of defective interfering rubella virus particles

Rubella virus (RV) particles produced under varying degrees of autointerference have been studied. RV has been purified from these stocks and studied by isopycnic centrifugation. RNA was extracted from the purified virions, labeled with ¹²⁵I, electrophoresed on 5% polyacrylamide slab gels, and subjected to autoradiography. The viral particles varied considerably with respect to their density and their RNA content. Virions present in low-interference stocks were contained in one band at ? = 1.19 g/ml³. Two single-stranded RNA species could be extracted from this band with molecular weights of 2.95 and 2.80 × 10⁶. Virions present in high-interference stocks were contained in at least three bands with densities of 1.19, 1.17, and 1.15 g/ml³. The molecular weights of the RNA molecules extracted from these three bands were 2.95, 2.80, 1.25, and 1.05 × 10⁶. Therefore, defective RV particles have been detected which possessed a density less than 1.19 g/ml³, contained a smaller RNA, and elicited autointerference.     

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.     

Cucumber mosaic virus contains a functionally divided genome

The four main RNA species isolated from purified Q strain of cucumber mosaic virus (CMV) were separated on 2.4% polyacrylamide gels. Using 16S and 23S Escherichia coli ribosomal RNA as molecular weight standards, the molecular weights of species 1–4 of CMV RNA were estimated in aqueous gels to be 1.30 × 10⁶, 1.13 × 10⁶, 0.78 × 10⁶, and 0.34 × 10⁶ daltons and in gels run in 98% formamide to be 1.35 × 10⁶, 1.16 /sx 10⁶, 0.85 × 10⁶, and 0.35 × 10⁶ daltons. The three largest species were essential for infectivity as determined by local lesion assay on cowpeas (Vigna sinensis). The variation of infectivity with the concentration of CMV and of CMV RNA has shown single hit kinetics even though lesion formation probably requires the initial simultaneous infection of each cell by the three largest RNA species.     

Defective interfering particles of Semliki Forest virus generated in BHK cells do not interfere with viral RNA synthesis in Aedes albopictus cells

Defective interfering (DI) particles of Semliki Forest virus (SFV) were generated by serial high multiplicity passage of plaque-purified virus on BHK cells. Defective interfering passages of SFV depressed the synthesis of 42 and 26 S viral RNA and induced the formation of two new single-stranded RNA forms (molecular weight, 1.2 × 10⁶ and 0.56 × 10⁶) in BHK and Vero cells but not in Aedes albopictus cells. These results suggest that the invertebrate cells restricted replication of the alphavirus DI particles.     

Replication of tobacco ringspot virus. II. Differences in nucleotide sequences between the viral RNA components

Tobacco ringspot virus (TRSV)-specific double-stranded (ds)-RNA was used in competition hybridization experiments to compare the nucleotide sequences of the viral RNAs. The results indicate that the RNA of molecular weight 1.4 × 10⁶ (RNA₁) isolated from either middle or bottom component particles of TRSV have indistinguishable nucleotide sequences. However, the sequences of the RNA of molecular weight 2.3 × 10⁶ (RNA₂) are distinct from those of RNA₁ although these RNAs may have sequences of about 900 nucleotides in common.     

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.     

The presence of common host sequences in different populations of substituted SV40 DNA.

Ribonucleic acids (RNAs) of turnip mosaic (TurMV), tobacco etch (TEV), and maize dwarf mosaic viruses (MDMV) have molecular weights of 3.31 × 10⁶, 3.14 × 10⁶, and 2.89 × 10⁶ determined by polyacrylamide gel electrophoresis, and of 3.07 × 10⁶, 3.33 × 10⁶, and 2.94 × 10⁶ determined by sedimentation analysis in linear log sucrose density gradients. These correspond to S values of 39.0, 40.6 and 38.2. When denatured by formaldehyde, molecular weights are TurMV, 3.07 × 10⁶; TEV, 3.04 × 10⁶; and MDMV, 2.90 × 10⁶ determined by gel electrophoresis. Sedimentation coefficients after denaturation are TurMV, 24.7 S; TEV, 24.9 S; and MDMV, 24.2 S, corresponding to molecular weights of 3.16 × 10⁶, 3.32 × 10⁶, and 3.06 × 10⁶ for TurMV, TEV, and MDMV, respectively. The reduction in electrophoretic mobility and sedimentation coefficient after denaturation, along with hyperchromicity and T_m measurements, suggests that all three virus RNAs have a similar degree of secondary structure.     

Further studies on Chara corallina virus

The particles of Chara corallina virus (CCV) and those of tobamoviruses share many properties, but differ in some. CCV particles are tubular, 532 nm long, and 18 nm wide, and are helically constructed with a basic pitch of 2.75 nm; their isoelectric point is pH 3.4–3.7; their sedimentation coefficient (s₂₀, w) is 230 S; they contain 5% RNA with a molecular weight of 3.6 × 10⁶ daltons, and with a base ratio of G 24.5, A 28.0, C 20.0, U 27.5; their coat protein is similar in size to that of tobamoviruses (about 17.5 × 10³ daltons). CCV is considered to be a tobamovirus. CCV may be transmitted experimentally to uninfected Chara corallina cells by injection of virus particles, and causes chlorosis and death in about 10–12 days. A classification of CCV and 68 other tobamovirus isolates, whose coat protein composition is known, showed that CCV is distant from all, but is most closely related to the cucurbit tobamoviruses.     

Studies on the in vitro uncoating of poliovirus. III. Roles of membrane-modifying and -stabilizing factors in the generation of subviral particles

A quantitative assay for defective-interfering (DI) particles of Sindbis virus (SV) was developed and shown to have an efficiency comparable to that of the plaque assay for infective virus. The assay was used to quantitate the kinetics of ultraviolet inactivation of the interfering activity of DI particles. For SV_BP19 (a virus stock produced by 19 serial undiluted passages in BHK cells) and SV_A17 (produced by 7 serial undiluted passages in A. albopictus cells) the interfering activity was seven- and twofold, respectively, more resistant to inactivation by uv light than was infectivity. For the DI particles in SV_BP19 the uv target size correlated very well with the size of the DI genome RNA (MW = 0.59 and 0.63 × 10⁶) but for the DI particles in SV_AP7 the uv target size was 25–30% smaller than the DI genome (MW = 3.2 × 10⁶). These results suggest that the entire genome of the DI particle in SV_AP7 may not be required for interference. In contrast to the results obtained with DI particles, the interfering capacity of a nondefective ts mutant was almost as sensitive to inactivation as was infectivity.