Equine herpesvirus in vivo: cyclic production of a DNA density variant with repetitive sequences.

The DNA of a strain of equine herpesvirus type 1 passed more than 500 times in Syrian hamsters (EHV-1ha) has been analyzed by CsCl equilibrium density gradient ultracentrifugation, analytical sedimentation, and DNA-DNA reassociation kinetics. The viral DNA consisted of light and heavy species having densities in CsCl of 1.716 and 1.724 g/cm³, which correspond to guanine plus cytosine contents of 56 and 64%, respectively. These values were confirmed by T_m measurements. Similar molecular weight values were obtained by analytical sedimentation for the light (87.9 × 10⁶) and heavy (81.8 × 10⁶) DNA species. The heavier species was produced in a cyclic manner. Hamsters infected with virus containing a high proportion of the heavy species gave reduced virus yields and survived longer. The genetic relatedness of the two viral DNA species of EHV-1ha was compared by examining the ability of each to reanneal with ³²P-labeled viral DNA of the tissue culture strain (L-M cell) of EHV-1 (EHV-1tc). The lighter (1.716 g/cm³) species of EHV-1ha was composed of unique sequences completely homologous to the entire EHV-1tc genome, while the heavier species (1.724 g/cm³) consisted of sequences homologous to approximately 50% of the EHV-1tc genome. Of these homologous sequences, 40–60% (20–30% of the entire EHV-1tc genome) were reiterated. Further, analyses of the EHV-1tc genome (fragmented and unfragmented) by thermal chromatography on hydroxylapatite and in neutral preparative CsCl equilibrium density gradients revealed considerable intramolecular heterogeneity in nucleotide distribution. Finally, analysis of the structural polypeptides of virions of EHV-1ha which contained the heavy and light DNA species revealed that the following two major viral proteins were missing from virions containing the heavier DNA species: VP8, an envelope protein with a molecular weight of 173,000, and VP23, a nucleocapsid protein with a molecular weight of 38,000.     

The polypeptide composition of avian infectious bronchitis virus particles

Summary Egg grown avian infectious bronchitis virus (IBV) centrifuged on sucrose density gradients was found to consist of a major virus peak of density 1.17 to 1.18 g/cm³ and occasionally two minor virus peaks of density 1.21 to 1.22 g/cm³ and 1.13 g/cm³. Three different IBV strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. The polypeptides of the different density virus particles from the three IBV strains were analysed on polyacrylamide gels. In all cases 7 polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. The polypeptides have been called VP1 (molecular weight 130,000), VP2 (105,000), VP3 (97,000), VP4 (81,000), VP5 (74,000), VP6 (51,000) and VP7 (33,000). Additional polypeptides were produced if slightly harsher treatments were used.With 4 Figures     

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.     

Effect of physical and chemical treatment on the buoyant density of reovirus

Summary Reovirus type 2 was treated with genetron or chloroform, heat (52° to 80°C), trypsin, and combinations of heat and trypsin. The treated virions were analyzed in a CsCl density gradient. Genetron and chloroform had no effect on the buoyant density of the virion, heat treatment resulted in a virus particle with a lowered density (1.35 g/cm³), and trypsin treatment rendered virus cores with a density of 1.42 g/cm³. When heat-treated virus was treated with trypsin, the particles also had a density of 1.42 g/cm³; however, trypsinized particles became more dense (1.46 g/cm³) when heated after the enzyme treatment.     

Subfractionation of CsCl-purified H-1 parvovirus on metrizamide gradients.

The different density classes of H-1 parvovirus, collected within 30 hr of parection of parasynchronous cultures, following the standard CsCl purification step, have been shown to be heterogeneous. Rebanding of the denser form (HF, ? = 1.46 g/cm³) and the less dense form (LF, ? = 1.42 g/cm³) of infectious virus in the nonionic density generating solute, metrizamide, showed that both HF and LF virus bands were heterogeneous in density. The infectivity banded with isotopically labeled virus protein and DNA at 1.32 g/cm³ for both HF and LF virus. Amounts of protein and DNA which varied from preparation to preparation, but which were greater from the HF virus band, were distributed throughout the rest of the gradient, but predominated in a peak at a density of 1.2 g/cm³. The protein in this peak was without hemagglutinating activity but had the molecular weights and proportions of the H-1 virion proteins (VP1, VP2′, and VP2). The DNA was of the same size as H-1 DNA monomers and its proportion to the protein was similar to that of the infectious peak. The DNA was susceptible to micrococcal nuclease digestion. The nature of this noninfectious viral material thus seemed to be incompletely assembled virus. Radio-labeled H-1 virus collected after 72 hr of infection formed a discrete single peak in both CsCl (? = 1.42 g/cm³), and metrizamide gradients (? = 1.32 g/cm³). There was no significant amount of the 1.20 g/cm³ viral protein-DNA complex in these mature preparations.     

Proteins specified by African swine fever virus

Summary At least 28 polypeptides have been identified in intracellular virus, with molecular weights ranging from 11,500 to 243,000 daltons. By treatment with Nonidet P-40 and 2-mercaptoethanol it is possible to obtain subviral particles that have lost some proteins and have a density in CsCl of 1.31 g/cm³ which is higher than that of the complete virus (1.23 g/cm³). After addition of NaCl the virus loses its major protein VP73 which indicates that it is localized in the viral envelope. Cores obtained after this treatment are made up of at least 14 proteins. Incorporation of³H-fucose and³H-glucosamine in intracellular virus occurs in three minor components. The protein VP42 is possibly the cell actin and appears to be strongly associated with the virus. It is not possible to eliminate it under conditions where the viral envelopes desappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.With 5 Figures     

Heterogeneity of infectious bronchitis virus grown in eggs

Summary Egg-grown infectious bronchitis virus, strain Beaudette, was concentrated and centrifuged on sucrose density gradients to separate the virus into five peaks with densities of 1.144, 1.160, 1.172, 1.191 and 1.218 g/cm³. All peaks retained infectivity, complement fixation activity and were labelled with³H-uridine. Morphologically the densest peak consisted of very large virus particles and amorphous material, the other peaks consisted of mainly intact particles although small differences in size and pleomorphism were seen.Polyacrylamide gel electrophoresis of material from the density gradient peaks revealed four major polypeptides and at least 10 minor polypeptides. The proportions of the polypeptides were approximately similar for all peaks with the exception of the densest peak in which the major polypeptides were greatly reduced. The four major polypeptides had approximate molecular weights of 1. 52,000, 2. 45,000, 3. 34,000, 4. 32,000. The major polypeptides 1 and 4 were shown to be glycosylated as were two of the minor polypeptides.With 8 Figures     

Cell-free assembly of a polyoma-like particle from empty capside and DNA.

A polyoma-like particle (PLP) is formed when polyoma DNA and purified empty capsids are incubated in a cell-free system. The DNA of this new particle is protected against the action of pancreatic DNase. The density of the purified PLP in CsCI is 1.32 g/cm³, which is intermediate between that of polyoma virions (1.34 g/cm³) and empty capsids (1.29 g/cm³). Purified PLP sediments at 190 S in sucrose and is stable in solutions of high ionic strength. When the DNA is extracted from PLP by the use of detergent and phenol, it is found to be doublestranded with a molecular weight of approximately 1.1 × 10⁶. The particles are stable in CsCl at 4° for at least 5 months. Electron micrographs indicate that highly purified PLPs stained with 2% PTA have the same appearance as polyoma capsids. Neither aggregates nor complexes bound by loose ionic bonds appear reasonable to explain these results. The evidence indicates that the DNA of this new polyoma-like particle, made under cell-free conditions, is protected by the capsid.     

Biochemistry of Theiler''s murine encephalomyelitis virus isolated from acutely infected mouse brain: identification of a previously unreported polypeptide

The WW strain of Theiler's murine encephalomyelitis virus (WW-TMEV) was purified from homogenates of acutely infected mouse brain. Infectious WW-TMEV was found to have an estimated sedimentation coefficient of 156 (s20,w) and a density of 1.35 g/cm3 in CsCl. Electron microscopy revealed a homogeneous population of 26-nm nonenveloped particles. Iodination of sodium dodecyl sulfate (SDS)-disrupted virions revealed four major capsid proteins with molecular weights of 58,000, 37,000, 34,000, and 27,000. A 6,000-dalton polypeptide was observed after long exposures of autoradiograms. The 37,000-, 24,000-, 27,000-, and 6,000-dalton polypeptides corresponded to picornaviral VP1, VP2, VP3, and VP4 capsid polypeptides, respectively. Comparison of autoradiograms of virions radiolabeled before and after SDS disruption indicated that the 58,000-dalton protein, VP2, and VP3 preferentially bound 125I under the labeling conditions used. Direct evidence was obtained that VP2 and VP3 were derived from the 58,000-dalton polypeptide by isolation of the 58,000-dalton polypeptide from polyacrylamide gels run under nonreducing conditions and subjecting it to reelectrophoresis under reducing conditions. The effect of trypsin on purified virions and their polypeptides was also investigated. Trypsin-sensitive sites were found in the 58,000-dalton protein, VP1, and VP2. Our results indicate that, in addition to the four typical picornaviral capsid polypeptides, there is a 58,000-dalton polypeptide present in WW-TMEV, which is sensitive to trypsin and can be reduced into two of the capsid proteins, VP2 and VP3.     

Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus

Summary The structural polypeptides of the isolate VR-2332 of porcine reproductive and respiratory syndrome virus were analyzed in sucrose gradient-purified virions. The virus had an average density of 1.15 g/cm³ and contained, by SDS-PAGE, three major polypeptides with apparent molecular weights of 15, 19 and 26–30 kDa, which were designated as nucleocapsid (N), matrix (M) and envelope (E), respectively. The predominant structural protein was N. N-glycosidase F digestion only affected E whereas O-glycosidase or endoglycosidase H digestion had no effect, suggesting that the viral glycoproteins contain only complex N-linked carbohydrates.     

The genomic RNA of mengovirus. II: Comparative analysis of the standard and denser virions.

The standard Mengovirus particle (buoyant density, 1.34 g/ml) was separated from a denser one (1.44 g/ml) by isopycnic centrifugation in CsCl at pH 8.0. When sedimented through a neutral sucrose density gradient, the standard virions had an apparent sedimention coefficient of 190 S, whereas the denser particles were resolved into two unequal peaks at 220 and 190 S. The denser particles were infectious, and the proportion of denser to standard virions remained unmodified after 24 serial passages at a high m.o.i. The progeny of both the standard and denser particles contained the same relative proportion of standard and denser virions. ³²P-labeled RNA extracted from denser particles was degraded to a variable extent. However, all of the big T₁ oligonucleotides characteristic of the standard Mengovirus genome were also present in the fingerprints of the RNA from the denser particles. These two findings suggested that the RNAs of the standard and denser particles were indeed the same, but that the general configuration of the denser virions made their RNA more accessible for interaction with the cesium salt and more susceptible to nuclease degradation. Analysis by SDS-PAGE of the ³⁵S-labeled structural proteins revealed that the denser virions lacked a polypeptide with an apparent MW of 59,000. The remaining polypeptides were present, although their relative proportions were somehow altered. The significance of the denser particles is discussed.     

A structural model for picornaviruses as suggested from an analysis of urea-degraded virions and procapsids of coxsackievirus B3

Urea treatment (3 M, 15 min, 37 °C, pH 9) of coxsackievirus B3 inactivated virus infectivity and degraded the virus capsid into substructures recoverable on sucrose gradients. One substructure which sedimented around 20 S contained VP1 and VP3, the other substructures which sedimented at 5 S contained VP2 and VP4, respectively, as analyzed by SDS polyacrylamide electrophoresis. The VP2 and VP4 polypeptides in the 5 S peak were probably separate since their molar ratios differed over the peak, and VP4 could be removed by dialysis. Treatment of the virions with only 1 M urea for 5 min yielded four peaks of radioactivity on sucrose gradients which sedimented at about 150 S (undegraded virions), 75–80 S (capsids minus VP4), and the 20 S and 5 S structures referred to above, suggesting a stepwise degradation of B3 virions by urea. The procapsids also were degraded into substructures which were separated on sucrose gradients; one sedimenting at around 40 S containing mostly VPO, and the other sedimenting around 20 S containing only VP1 and VP3. When coxsackievirus B3-³⁵S cysteine-labeled virions were disrupted and analyzed on SDS gels, all polypeptides except VP4 were labeled, suggesting that VP2 and VP4 are distinct polypeptides. Analysis of urea-disrupted coxsackievirus B3 substructures provides the basis for a T = 3 structural model of the picornaviruses, with 12 pentamers (VP2 and VP4) and 20 hexamers (VP1 and VP3) per virion.     

Respiratory syncytial virus proteins

Respiratory syncytial (RS) virus grown in BS-C-1 cells was concentrated from the fluid of infected cultures by precipitation with polyethylene glycol (PEG) and banded by isopycnic centrifugation in sucrose or metrizamide density gradients. At least six virus-specified polypeptide bands, one of which was heterogeneous, could be resolved by continuous SDS-polyacrylamide gel electrophoresis (PAGE) and an additional band by discontinuous SDS-PAGE. The three predominant viral polypeptides were a glycopolypeptide of 48 × 10³ (VGP48), a nucleocapsid polypeptide of 41 × 10³ (VP41), and a polypeptide of 27 × 10³ molecular weight (VP27). Three minor viral polypeptides have been assigned the molecular weight of 38 × 10³ (VP38), 32 × 10³ (VP32) and 25 × 10³ (VP25). A minor glycopolypeptide of molecular weight 42 × 10³ (VGP42) may exist also. Partial purification was accompanied by the loss of high molecular weight glycopolypeptides; however, one high molecular polypeptide (P2) remained consistently associated with the presumptive polypeptides and may represent an eighth virus-specified polypeptide.VP27 can be obtained in relatively pure form by sedimentation of detergent-treated RS virus in a metrizamide gradient containing detergent.     

Characteristics of venezuelan equine encephalomyelitis virus ribonucleoprotein

Summary Venezuelan equine encephalomyelitis (VEE) virus grown in chick embryo fibroblasts was concentrated by differential centrifugation and purified in sucrose and caesium chloride density gradients. Treatment of the virus with Tween 80 and ether appeared to be the optimal method for obtaining biologically active subviral components. In experiments with sucrose density gradient centrifugation the sedimentation coefficient of the virus was about 380 S and that of virus ribonucleoprotein (RNP) was about 160 S. In experiments with caesium chloride equilibrium density centrifugation virions banded at the buoyant density of 1.25 g/cm³ and virus RNP banded at 1.42 g/cm³.     

Defective virions of human cytomegalovirus.

Passage of human cytomegalovirus at high multiplicity of infection generated defective virus particles which banded in CsCI at a lower buoyant density than standard virus. The DNA from defective virions banded at a lower buoyant density in CsCl than standard DNA and sedimented in sucrose gradients similar to standard DNA. Contour length measurements of DNA from defective virions revealed various classes of DNAs ranging in molecular weight from 40 to 120 x 10⁶; this was in contrast to standard DNA with a molecular weight of 150 x 10⁶.     

Comparison of influenza and parainfluenza RNP properties

Summary Fowl plague virus and Sendai virus were disrupted by Tween 20 in an alcaline medium. Whereas in the case of fowl plague virus sedimentation of the ribonucleoprotein (BNP) in sucrose gradients revealed three components sedimenting at 64S, 58S, and 48S, the RNP of Sendai virus showed a sedimentation coefficient of 110S only. During buoyant density studies in CsCl the three components of fowl plague virus RNP banded at the uniform density of 1.35–1.36 g/cm³ and the RNP of Sendai virus was found at a medium density of 1.32 g/cm³.     

Identification of point mutations in the genome of the poliovirus Sabin vaccine LSc 2ab, and catalogue of RNase T1- and RNase A-resistant oligonucleotides of poliovirus type 1 (Mahoney) RNA

In harvests of a feline calicivirus (FCV) grown in feline embryo cells two populations of viral components were observed after centrifugation in sucrose and CsCl gradients. Particles in the first (PI) were mature virions since they had a buoyant density of 1.39 g · cm^?3, a sedimentation coefficient of 170 S, were infectious and showed characteristic calicivirus morphology by electron microscopy. The second particle (P2), which in terms of protein was 10 times as abundant as P1, had a buoyant density of 1.31 g · cm^?3, a sedimentation coefficient of 15 S, and contained little or no infectivity. Evidence that P2 was virus specific was that P2 as well as P1 contained a polypeptide with a molecular weight of 65,000 which is the weight of the viral capsid protein, and P1 and P2 shared an antigenic determinant which was responsible for inducing neutralizing antibodies. The 15 S component was not generated from the virion by conditions encountered during viral growth and purification. It is considered likely that the 15 S particle is a stable product of FCV infection and may be a subunit in the assembly of the viral capsid.     

Investigation of the structure of polio- and human rhinovirions through the use of selective chemical reactivity.

The configurations of poliovirus and human rhinovirus type 2 (HRV-2) virions and subviral particles were investigated by measuring the relative accessibility of the four virion polypeptides (VP1-4) to the labeling reagents [³H]acetic anhydride, ¹²⁵I, and [¹⁴C]iodoacetamide. The reaction of [³H]acetic anhydride with the intact virions of poliovirus or HRV-2 revealed that in both cases VP1 was labeled to a greater extent than the smaller polypeptides, VP2 and VP3. The smallest polypeptide, VP4, was not labeled at all, suggesting that it may be internal and may not contribute directly to the surface properties of native virions. Treatment of poliovirus at 47° or HRV-2 at pH 5 produces slower sedimenting A-particles which lack VP4 and resemble the particles produced during the early interaction of virus with host cells. The occurrence of a configurational change in the formation of A-particles was suggested by the observation that A-particles exhibited increased relative susceptibility to labeling of VP2 with [³H]acetic anhydride. Virions which had been disrupted by treatment with dodecyl sulfate at 100° were labeled with [³H]acetic anhydride approximately uniformly in all polypeptides including VP4. The labeling patterns of the polypeptides of poliovirions, A-particles, and disrupted virus with ¹²⁵I were qualitatively similar to those obtained with [³H]acetic anhydride. In contrast, VP1 in HRV-2 virions was relatively protected from labeling with ¹²⁵I and became accessible only in A-particles or disrupted virions. With both viruses, again, VP4 was not labeled with ¹²⁵I in intact virions but was labeled after virus disruption. Very little [¹⁴C]iodoacetamide was incorporated into native virions, although in a few experiments VP2 was labeled in HRV-2. Only the capsid polypeptides VP1, VP2, and VP3 were labeled with [¹⁴C]iodoacetamide following disruption with SDS, indicating that neither the poliovirus VP4 nor the HRV-2 VP4 contain free SH groups.     

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.     

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.