- Volume 64, Issue 4, 1983

A drastic modification in permeability to monovalent ions occurred in HeLa cells infected with poliovirus, starting in the period from the third to the fourth h post-infection. The bulk of poliovirus protein synthesis took place from the third to the sixth h in cells in which the concentration of monovalent ions in the cytoplasm had changed considerably compared to uninfected cells. Under our conditions of infection (HeLa cells grown in monolayer), poliovirus translation continued beyond the eighth h. Modification of permeability to 86Rb+ ions induced by poliovirus infection was prevented by the addition of 2 mm-guanidine at the time of infection. However, when poliovirus replication was allowed to take place for 1 h before addition of guanidine, the membrane did become modified to some extent. The degree of leakiness to 86Rb+ ions increased when guanidine was added later. The blockage of membrane leakiness by early addition of guanidine was overcome by the addition of choline. The inhibition of cellular protein synthesis, which occurred early in infection or in the presence of guanidine, did not coincide with the modification of permeability to 86Rb+ ions. Viral protein synthesis was necessary in order to modify the membrane late in infection, since addition of 10−5 m-cycloheximide during the first 2 h of infection prevented the leakiness to 86Rb+ ions observed from the fourth h after infection. Membrane potential, as measured by the lipophilic cation TPP+ (tetraphenylphosphonium), dropped from the fourth h post-infection. This change in the membrane was also prevented when viral gene expression was inhibited by the presence of guanidine.

The mechanism of action of a new anti-rhinovirus compound, dichloroflavan, was examined using a variety of techniques. Dichloroflavan was shown to bind to the highly sensitive rhinovirus type 1B (50% inhibitory concentration 0.007 µmm) and at a much lower level to the insensitive rhinovirus type 4 (50% inhibitory concentration > 25 µm). Binding of the compound to rhinovirus 1B was accompanied by a reduction (about 0.5 log10 units) in virus infectivity which could be restored by treatment with chloroform. Maximum inhibition of virus yield (about 2 log10 units) occurred only when compound and virus were added together, but some inhibition (about 0.7 log10 units) occurred even when the compound was added near the end of a single cycle of replication. This late reduction in infectivity could be abolished by treating with chloroform, and was therefore caused by the compound binding to progeny virus. The compound did not interfere with adsorption of virus to cells, nor with uncoating of the viral RNA. However, little or no viral RNA and protein synthesis occurred in the presence of dichloroflavan provided that the compound was added with the virus. Addition of the compound after virus adsorption had no effect on either viral RNA or protein synthesis. These results indicate that dichloroflavan binds directly to rhinovirus 1B and appears to prevent virus replication at a point immediately after uncoating of the viral RNA.

The role of antibody and cell-mediated immunity in the resistance to Japanese encephalitis virus (JEV) infection was studied in adult mice. Passively transferred antibodies obtained up to 2 weeks after primary infection protected the recipient mice against a challenge infection with JEV. Antibody obtained at 4 or 5 weeks failed to protect despite the presence of high titres of neutralizing antibody. Protection was abrogated by pretreatment of the early serum with 2-mercaptoethanol to remove IgM. Similarly, adoptive transfer of immune spleen cells obtained up to 2 weeks after immunization provided protection. The protective effect was abolished by pretreatment of the immune spleen cells with anti-Thy 1.2 antiserum and complement. These findings suggest a role of T lymphocytes and IgM antibody in recovery from JEV infection.

Sendai virus grown in LLC-MK2 cells is known to have low infectivity, no detectable haemolysing ability and predominantly uncleaved F glycoprotein. Treatment of such virus with chicken amniotic fluid resulted in a 103- to 104-fold increase in infectivity, the development of haemolysing ability, and cleavage of the F glycoprotein. The ‘Sendai activating enzyme’ (SAE) responsible for these effects required the presence of 1 mm-Ca2+ ions for maximum activity. It was inhibited by phenylmethylsulphonyl fluoride and soybean trypsin inhibitor but was unaffected by sulphydryl-blocking agents. The results of gel filtration tests suggested that the molecular weight of SAE was about 55000. SAE may be the natural proteolytic activator of Sendai virus in a soluble form.

Four proteins, GP1, VGP48, GP26 and VPM27, are associated with the envelope of respiratory syncytial (RS) virus. The status of GP1 has been uncertain, because a cellular glycoprotein migrates at the same position when Laemmli’s discontinuous buffer system is used for PAGE, and because BSC-1 cells infected with the RSN-2 strain of RS virus appear not to contain GP1. However, additional evidence suggests that GP1 is a viral structural protein. (i) It is removed from cells by trypsin, while the cellular glycoprotein is not; (ii) it is separated from the cellular glycoprotein when the infected cells are analysed by neutral SDS-PAGE; (iii) it is present in the purified RSN-2 strain of RS virus produced by BSC-1 cells; (iv) it is also present in the purified Long strain of RS virus produced by either human or monkey cells. When purified Long strain virus is analysed by PAGE under non-reducing conditions, the glycoproteins VGP48 and GP26 migrate together, and VPM27 separates into two proteins, which one-dimensional peptide mapping suggests are not different proteins. These observations suggest that VGP48 and GP26 exist in the virion as a single molecule joined by disulphide bonds, and so resemble a paramyxovirus fusion protein, and that probably there are two forms of VPM27 which differ in either position or number of disulphide bonds.

The structure and organization of the ribonucleoprotein (RNP) complex of an arenavirus, Pichinde virus, was investigated. The basic configuration of the RNP was found to be a linear array of globular subunits or nucleosomes, 4 to 5 nm in diameter, that represent individual molecules of the major N polypeptide. This filament appears to fold progressively through a number of intermediate helical structures, 12 to 15 nm in diameter, that reveal an increasing number of nucleosomes associated with each turn of the helix. They range from a fragile configuration of two or three nucleosomes per turn to a more stable fibre in which the nucleosomes cannot be resolved. The strands were shown to form closed circles and it appeared that the twisting of these circular forms resulted in the formation of 20 nm-thick fibres which were seen in isolated viral core structures. The association of these RNP structures with other viral components is discussed.

Antigenic variation in the glycoprotein of rabies (CVS-11) virus was studied. Neutralization-resistant variant viruses were isolated in vitro at high frequency (10−4 to 10−5) in the presence of anti-glycoprotein monoclonal antibody. Analysis of these variants identified at least three functionally independent antigenic sites, based on the grouping of variants that were no longer neutralized by one or more of a panel of 24 monoclonal antibodies. Competition radioimmunoassay suggested that one of these three antigenic sites was topologically distinct, with the other two in close proximity. In addition, it was shown that most (but not all) neutralization-resistant variants failed to bind the relevant monoclonal antibody. Viruses with altered antigenicity were shown to accumulate in virus stocks following several passages in vitro in the absence of antibody. In addition, variants were isolated in vivo following treatment of mice with monoclonal antibody.

Turkey and chicken rotaviruses were successfully adapted to replicate in a rhesus monkey embryo kidney (MA104) cell line. Trypsin treatment of virus and cells was essential for serial passage of the viruses. Polyacrylamide gel electrophoresis separated the viral RNA into 11 segments with clear resolution of segments 10 and 11. Segment 5 migrated with size class I, and this characteristic appeared to be a unique feature of the avian rotavirus genome. The ability of the avian rotaviruses to haemagglutinate a variety of erythrocytes was demonstrated. A type-specific antigen was detected by haemagglutination-inhibition assays and a group or subgroup antigen by enzyme immunoassays. Treatment of serum with heparin-MnCl2 was shown to be the best method for removing non-specific inhibitors of haemagglutination.

Three types of regularly constructed assembly have been observed by electron microscopy of rotavirus-containing faeces from naturally infected cattle. Two of the structures, designated rotatube 1 and rotatube 2 respectively, are tubular in nature, and the third, designated rotatube 3, takes the form of large relatively disorganized sheets of material exhibiting, in some areas, tubular characteristics. Rotatube 1 is a ‘wide’ tube of approximately 80 nm diameter, the wall of which is composed of a surface lattice of hexagonally arranged subunits. Rotatube 2 is a ‘narrow’ tube, half the diameter of the wide tube, and does not exhibit a strictly hexagonal surface lattice. The sheets of rotatube 3 material are composed of subunits arranged on an hexagonal lattice identical with that of rotatube 1. The dimensions and general structural characteristics of the three types of assembly indicate that they are composed of rotavirus capsid protein.

The number of discrete species of glycoprotein induced by herpes simplex virus type 1 (HSV-1, strain 17 syn+) and their processing has been examined by a combination of immunoprecipitation with monoclonal antibodies and analysis of immune precipitates by two-dimensional (2D) gel electrophoresis. Seventeen different monoclonal antibodies directed against glycoproteins A/B, C, D and E were used. Polypeptides intermediate in the synthesis of gA/B, C and D were visualized and two early intermediates of glycoprotein A/B (pgA/B1 and pgA/B2), both mannose-containing, were identified. Comparison on 2D gels of polypeptides synthesized in vitro from HSV-1-infected cell mRNA with those synthesized in pulse-chase experiments in vivo has allowed identification of early precursors of pgA/B and pgD. Their apparent mol. wt. were 105000 and 47000 respectively. The 2D gel analysis of glycoproteins induced in HFL cells infected with 17 syn+ revealed a number of previously unreported glycoprotein species. One had mobility on both gradient and single concentration SDS-polyacrylamide gels similar to that of gC but was resolved from gC on non-equilibrium pH gradient gels. This glycoprotein was produced in relatively large amounts and was not precipitated by any of the monoclonal antibodies used in this study. The data suggest that this glycoprotein either has a polypeptide chain unrelated to those of glycoproteins A/B, C, D or E or alternatively is derived from one of them and modified in such a way as to mask or remove the antigenic sites with which the monoclonal antibodies interact. Sixteen other previously unreported glycoprotein species not obviously related, as judged by their electrophoretic mobility, to gA/B, C, D or E were also identified: two reacted with gA/B-specific monoclonal antibodies and five with glycoprotein C-specific monoclonal antibodies. The origin of the remaining nine new glycoproteins has still to be ascertained.

The relative contents of Epstein—Barr virus (EBV)-associated nuclear antigen (EBNA) and average numbers of EBV genome equivalents per cell were determined (i) in Raji cells superinfected with P3HR-1 virus, (ii) in Raji cells induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium n-butyrate and (iii) in P3HR-1 cells induced by TPA and sodium n-butyrate. This treatment was followed by increases in the percentage of early antigen (in both Raji and P3HR-1 cells) and virus capsid antigen-positive cells (in P3HR-1 cells) and increases of approximately 20-fold in the average number of EBV DNA equivalents in superinfected Raji cells and in TPA- and sodium n-butyrate-induced P3HR-1 cells. However, the content of EBNA in these cells dropped. This was revealed by a decrease in both the complement-fixing antigen content and in the proportion of EBNA-positive cells as determined by anti-complement immunofluorescence. Thus, the positive correlation found previously between the content of EBNA and the number of EBV genome equivalents per cell in proliferating lymphoblastoid cultures does not seem to apply to the situation in either superinfected Raji cells or in P3HR-1 cells induced by TPA and sodium n-butyrate.

Hepatitis virus B e antigen subtypes HBeAg/1 and HBeAg/2 can be distinguished by a micro-Ouchterlony immunodiffusion assay and a solid-phase radioimmunoassay using serum IgG-associated HBeAgs and liver- and serum-derived IgG-free HBeAgs. The liver- and serum-derived HBeAg consisted of two different antigenicities, HBeAg/1 and HBeAg/2. Both the liver-derived HBeAg/1 and HBeAg/2 had mol. wt. of 30000 (monomer) and 90000 (trimer) and shared the same isoelectric point of 4.3 to 4.8. Approximately 80% of liver-derived HBeAg (mol. wt. 30000 and 90000) had HBeAg/1 activity and the remaining 20% was HBeAg/2. The serum-derived HBeAg/1 and HBeAg/2 were both associated with IgG to form heterogeneous moieties with apparent mol. wt. of 240000, 400000 and 540000 or were free, with mol. wt. 30000 (monomer) and 90000 (trimer). The serum HBeAg of mol. wt. 90000 was further differentiated into two molecular species, one trimer and the other associated with albumin. Large types of both HBeAg/1 and HBeAg/2 were recovered by isoelectrictric focusing at pH 5.5 to 7.5 (mol. wt. 240000), 5.7 to 8.0 (mol. wt. 400000) and 6.4 to 8.4 (mol. wt. 540000) respectively, while small types of both exhibited a narrower range of isoelectric points similar to liver-derived antigens. In most sera, antigenicity of half of the serum IgG-associated HBeAg was HBeAg/1 and the remaining 50% was HBeAg/2, whereas 80% of the free HBeAg had HBeAg/1 activity and the remaining 20% was HBeAg/2. These results indicate that HBeAg/1 and HBeAg/2, although antigenically distinct, are very similar physicochemically with respect to both mol. wt. and isoelectric point. Dissociation of the ‘large’ HBeAg/1 and HBeAg/2 moieties into three mol. wt. species (160000, 32000 and 16000) was achieved by 6-m-guanidine-HCl treatment followed by gel filtration through Sepharose 6B. The 160000 species was IgG, having both anti-HBeAg/1 and anti-HBeAg/2 activities. In addition, the lower mol. wt. polypeptides (32000 and 16000) exhibited both HBeAg/1 and HBeAg/2 activities. Therefore, we suggest that the IgG associated with the HBeAg molecule forms a true immune complex rather than a non-specific association between HBeAg and IgG. Furthermore, the 32000 mol. wt. polypeptide split into two species of 16000 mol. wt. when heated at 100 °C for 2 min. On the basis of these results, the 16000 mol. wt. polypeptide may be assumed to be the essential polypeptide bearing HBeAg/1 and HBeAg/2 activities in the liver and serum.

Eight hybridomas secreting monoclonal antibodies specific to human fibroblast interferon (IFN-β 1) were identified by a new screening procedure based on immune precipitation with a second antibody and recovery of interferon activity from the immune precipitate. Immunoadsorbents were prepared from these monoclonal antibodies and used for purification of IFN-β 1. Electrophoretically pure IFN-β 1, having a specific activity of 3 × 108 to 6 × 108 units/mg was obtained by a single passage of partially purified IFN-β 1 on these immunoadsorbents.

We have investigated the use of liposomes as carriers for the transfer of simian virus 40 (SV40) DNA into mammalian cells. The amount of DNA entrapped in liposomes was dependent on the input DNA concentration and lipid composition. DNA remained intact after liposome encapsulation and was resistant to deoxyribonuclease digestion. Combined transfer to and expression of liposome-entrapped SV40 DNA in monkey kidney cells was assayed by infectious plaque formation. Negatively-charged liposomes containing phosphatidylserine were more effective in DNA transfer and expression than neutral liposomes. The inclusion of carrier salmon sperm DNA inhibited liposome-entrapped SV40 DNA infectivity. Infectivity of liposome-entrapped DNA was directly related to both liposome DNA concentration and number of vesicles added. Liposome-entrapped SV40 minichromosome was 20-fold more infective than free minichromosome, but only 20% more infective than liposome-entrapped SV40 DNA. Thus, the presence of hyperacetylated histones on the DNA failed to enhance liposome-mediated DNA transfer appreciably. Incubation of cells with various modulators of endocytosis implicated the endocytotic pathway in the mechanism of liposome-mediated DNA transfer. These studies show that liposomes are suitable carriers for the introduction of viral DNA and chromatin into mammalian cells.

Two host range mutants of avian sarcoma virus (ASV) were isolated from a clone of the Bratislava 77 strain, subgroup C (B77-C). An HR− mutant was obtained by adaptation to duck cells and showed almost the same efficiencies of transformation (e.o.t.) and growth in cultures of chick embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF). An HR+ mutant showed lower e.o.t. and slower growth on DEF than on CEF. Characterization of these mutants and wild-type B77-C showed that (i) unlike the host range mutants reported previously, these mutants and wild-type B77-C have common antigenicity, (ii) the number of infective centres is almost the same for the two mutants and the wild-type, and so the adsorption and integration of these viruses are similar, and (iii) the content of viral RNA in DEF infected with HR− is increased and decreased in the case of HR+ compared with the wild-type.

The genomes of diabetogenic and non-diabetogenic variants of encephalomyocarditis virus were analysed by nucleic acid hybridization and RNA fingerprinting. Hybridization and thermal elution profiles failed to show any difference between the RNAs of the two variants, whereas fingerprinting of the T1-digested RNAs revealed at least one oligonucleotide, 20 to 25 nucleotides long, missing in the non-diabetogenic variant.

The preexistence of a cytoplasmic membrane complex in HEp-2 cells, induced by poliovirus when inhibited in its reproduction by guanidine, was a prerequisite for accelerated reproduction of superinfecting Mouse Elberfeld (ME) virus. Guanidine-inhibited poliovirus induced a membrane complex of 470S that was successively modified into a faster sedimenting membrane complex (up to 700S) by superinfecting ME virus and exploited for ME virus reproduction. The modified membrane complex was the site for ME virus-specific RNA polymerization characterized by the existence of in vivo and in vitro activity of ME virus RNA polymerase associated with the modified membrane complex. Proof of membrane-bound RNA polymerase and newly synthesized ME virus RNA including replicative intermediate led to the conclusion that superinfecting ME virus exploits the ‘poliovirus/guanidine’-induced complex as the site of action of its replication complex.