Evidence for restricted replication of rubella virus in rat glial cells in culture

Intracellular rubella virus (RV) polypeptide synthesis during a productive infection of murine fibroblasts (L2 cells) has been investigated using immune precipitation techniques. Four structural and three additional intracellular polypeptides (p75, p60, VP44, VP41, p30, VP24, and VP19) were observed following polyacrylamide slab gel electrophoresis and autoradiography. However, when normal rat glial (RG) cells were infected only five polypeptides could be observed (p75, p60, VP44, VP41, and VP19). No infectious RV could be detected in tissue culture medium from flasks of infected RG cells. Calculations of relative concentrations of intracellular RV polypeptides precipitated from L2 and RG cells indicated that undetectable amounts of p30 and VP24, diminished amounts of p60 and VP19 and more than twice as much p75 were precipitated from infected RG cells. These data indicate that there is restricted replication of RV in normal rat glia.     

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 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.     

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.     

Polypeptides of Moloney sarcoma-leukemia virions: their resolution and incorporation into extracellular virions

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using four different concentrations of acrylamide resolved 16 polypeptide and glycopeptide components of Moloney sarcoma-leukemia virions radiolabeled with amino acids and d-glucosamine. Virions labeled with one neutral (lecuine or tryptophan) and one basic (lysine) amino acid show a distinctive label incorporation ratio for polypeptides in clustered molecular weight groups. One low molecular weight polypeptide is lysine rich, but is not a true histone since it was found to contain tryptophan.Several distinctive patterns of incorporation of pulse labeled ([³H]leucine) polypeptides into virions were observed. For most virion polypeptides the highest amount of pulse label recovered per unit time in extracellular virions was from early chase intervals (0–30, or 30–60 min). During subsequent chase intervals (60–120, or 120–180 min), the amounts of ³H counts in these specific labeled polypeptides recovered in extracellular virus per unit time decreased exponentially. Pulse labeled copies of one low molecular weight polypeptide (16,000) and the two major virion glycoproteins (73,000 and 75,000) appeared in supernatant virions during early chase intervals (0–30 min) at a low level then increased in amount of label recovered per unit time during chase intervals of 30–60, and 60–120 min. A third unique incorporation pattern was observed for one low molecular weight polypeptide (18,000). Pulse labeled copies of this polypeptide were not detectable in supernatant virus during the first 30 min of chase, but appeared in increasing amounts recovered per unit time during chase intervals of 30–60, and 60–120 min.     

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.     

Estimate of absolute specific infectivity of wound tumor virus purified with polyethylene glycol

Purification of wound tumor virus (WTV) was made simpler and quicker and a much greater proportion of active virions were obtained in the purified preparations of this labile virus by precipitating it with polyethylene glycol (PEG) 6000 before using density gradient centrifugation. Adjustment of clarified extracts to pH 5.7 gave the best yield of total (active plus inactive) virus and the highest specific infectivity. An optimal NaCl concentration (0.3 m) was important in obtaining good separation of the virus from host components during subsequent zonal density gradient centrifugation and maximal yields. NaCl at 0.2 m yielded purified virus of somewhat greater specific infectivity. Maximal yields of total virus were obtained with the use of 4% PEG for 2 hr although 3% PEG gave virus of about 1.5 times greater specific infeetivity. A histidine-MgCl₂ buffer solution for resuspending virus precipitated by PEG resulted in at least 50% greater total virus yields than did a glycine-MgCl₂ solution. WTV purified under the optimal conditions determined in this investigation yielded 3–5 times more total virus and at least 50 times more infective virus than did the standard method used previously. The purified virus had an absolute specific infectivity of about 25–30%, that is, it contained about 2 or 3 inactivated virions to each infective one. Purified preparations containing about 3 × 10¹¹ total virions per ml could be quick-frozen and stored at ?80 ° for at least a year without detectable loss of infectivity.     

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.     

Studies of the molecular anatomy of the L-M cell strain of equine herpes virus type 1: proteins of the nucleocapsid and intact virion

The structural components of purified enveloped virions and of purified nucleocapsids of the tissue culture strain (L-M cell) of equine herpes virus type 1 (EHV-1L) were analyzed by discontinuous sodium dodecyl sulfate polyacryalmide gel electrophoresis. Enveloped virions were comprised of 28 structural proteins of average molecular weights ranging from 270,000 to 16,000. Twelve of the proteins exhibited molecular weights of greater than 100,000, and six of these were above 200,000. Utilizing radioactively labeled compounds (³H-glucosamine and ³H-choline), four glycoproteins, four lipoproteins, and nine glycolipoproteins were shown to be present in the virions. Purified nucleocapsids, isolated from nuclear extracts of infected L-M cells, contained five major structural proteins with average molecular weights of 148,000, 59,000, 46,000, 36,000, and 18,000. These five proteins comprised greater than 96% of total nucleocapsid protein, on the basis of radioactivity. The 148,000 MW protein (VP 9) accounted for approximately 65% of the total nucleocapsid protein and was the major structural protein of both nucleocapsids and intact virions. None of these proteins corresponded to glycoproteins or lipoproteins present in enveloped virions, indicating that glycoproteins and lipoproteins are components of the envelope. The remaining 4% consisted of eight structural proteins ranging from 140,000 to 30,000 MW and were judged to be minor structural components, reproducibly present in all preparations of nucleocapsids.     

Structural similarities of hog cholera virus with togaviruses

Summary Hog cholera virus grown in PK-15 cells was purified by centrifugation through a sucrose cushion followed by sucrose gradient centrifugation. Analysis of virus labeled externally with [³H]sodium borohydride on polyacrylamide gel electrophoresis revealed two glycoproteins, gp55 and gp46. A third structural polypeptide, p36, seems not to be glycosylated. The gp46 was also found in the virus-free supernatant of infected cells. It could be demonstrated by radioimmune precipitation of virus labeled with [³⁵S]methionine that all three polypeptides are specific for hog cholera virions. Electron microscopically hog cholera virus appeared as a spherical particle with a diameter of 42±8 nm. The virus particles frequently displayed a fringe of projections with a length of about 6–8 nm. The similarities of hog cholera virus with Alphaviruses and Flaviviruses are discussed.With 5 Figures     

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.     

Herpes simplex virus protein synthesis in the presence of 2-deoxy-D-glucose.

The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [¹⁴C]amino acids or [¹⁴C]glucosamine in the presence or absence of 2-deoxy-d-glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 mM), the major envelope glycoprotein (VP123, MW 123,000) labeled with [¹⁴C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [¹⁴C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [¹⁴C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose.     

Structural proteins of equine arteritis virus

Summary Polyacrylamide gel electrophoresis of purified and solubilized equine arteritis virus (EAV) revealed nine structural proteins. Two of these proteins with molecular weights of 15,000 (VP7) and 13,000 (VP8) daltons are considered as major. The molecular weights of the seven minor proteins ranged between 72,000 and 10,500 daltons.The core fraction of the virion was dissociated from the envelope fraction by sucrose gradient centrifugation of virus treated with the nonionic detergent Nonidet P40 and phospholipase C. The core fraction contained RNA and one major protein (VP8), whereas the envelope contained one major protein (VP7) and the seven minor proteins. Six of the nine proteins (VP1 through VP6) were labeled with¹⁴C-glucosamine and are thus glycoproteins. VP8, a nonglycoprotein associated with the core fraction, is apparently the nucleoprotein of EAV.     

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector

Summary.  For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 × 10³ HA units/ml from infected Bm5 cells, 2.1× 10⁵ HA units/larvae from infected larval fat body, and 1.6× 10⁶ HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines. Received December 29, 1998/Accepted August 19, 1999     

The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves

The fraction of a homogenate of cowpea mosaic virus (CPMV) infected leaves sedimenting at 1000g for 15 min was further divided by centrifugation on a discontinuous gradient consisting of layers of 60, 45, and 20% sucrose. It was possible to separate the nuclei and nuclear fragments from most of the chloroplasts.Hybridization with [³H]uridine labeled CPMV RNA and electron microscopy were used to detect CPMV-specific double-stranded RNA in different fractions of the gradient. By hybridization 70–90% of the double-stranded RNA was found in the so called chloroplast-fraction. By electron microscopy it was confirmed that double-stranded RNAs with lengths characteristic for double-stranded viral RNAs occurred predominantly in this chloroplast-fraction. The chloroplast-fraction also contained vesiculated membrane structures and amorphous electron-dense material, which are characteristic structures from CPMV-infected cells, suggesting that CPMV-specific double-stranded RNA might be associated with either or both of these structures rather than with the chloroplasts.     

Rhinovirus species and clinical characteristics in the first wheezing episode in children

The clinical data on the first wheezing episodes induced by different rhinovirus (RV) species are still limited. We aimed to investigate the prevalence of RV genotypes, sensitization status, and clinical characteristics of patients having a respiratory infection caused by either different RV species or other respiratory viruses. The study enrolled 111 patients (aged 3–23 months, 79% hospitalized, 76% with RV infection) with the first wheezing episode. RV‐specific sequences were identified by partial sequencing of VP4/VP2 and 5′ non‐coding regions with 80% success rate. The investigated clinical and laboratory variables included atopic characteristics and illness severity, parental atopic illnesses, and parental smoking. Of the study children, 56% percent had > 1 atopic characteristic (atopy, eczema and/or blood eosinophil count > 0.4 × 10⁹/L) and 23% were sensitised to allergens. RV‐C was detected in 58% of RV positive samples, followed by RV‐A (20%) and RV‐B (1.2%). Children with RV‐A and RV‐C induced wheezing were older (P = 0.014) and had more atopic characteristics (P = 0.001) than those with non‐RV. RV‐A and RV‐C illnesses had shorter duration of preadmission symptoms and required more bronchodilator use at the ward than non‐RV illnesses (both P < 0.05, respectively). RV‐C is the most common cause of severe early wheezing. Atopic and illness severity features are associated with children having RV‐A or RV‐C induced first wheezing episode rather than with children having a non‐RV induced wheezing. J. Med. Virol. 88:2059–2068, 2016. © 2016 Wiley Periodicals, Inc.

     

Biochemistry and pathogenicity of echovirus 9 I. Characterization of the virus particles of strains Barty and Hill

Strain Barty, a mouse-pathogenic echovirus 9, which is known to cause epidemic disease in man, and strain Hill, the prototype of echovirus 9, were characterized and compared. Strain Barty virion particles sediment in a low-salt-sucrose gradient with 156 S, whereas particles of the Hill strain form fast-sedimenting aggregates. Plaque formation of the Hill strain is inhibited under an overlay containing agar; virus strain Barty, however, forms plaques. Isoelectric focusing revealed that Barty virion particles exist essentially in one conformation with a characteristic isoelectric point; virions of the Hill strain were found to behave like a mixture of particles with various isoelectric points. Thus, the two echovirus 9 strains, apparently, have a different capsid surface. These divergent capsid surface structures include not only the adsorption site(s)-strain Barty adsorbs less efficiently to cells in culture than strain Hill-but also the determinants inducing neutralizing antibodies: serologically, the two strains are clearly distinguishable. The capsid protein patterns of the two strains in SDS-PAGE show a characteristic difference: VPl of strain Barty is a double band, whereas strain Hill contains a single band in a position just between the two VP1 bands of strain Barty. In addition, fingerprinting to the capsid proteins revealed that the two echovirus 19 strains differ not only in VP1 but probably also in VP3. These determinants of capsid proteins may cause the observed divergent behaviors of the two virus strains, and-possibly-influence pathogenicity.