- Volume 25, Issue 2, 1974

Treatment of T3 with various denaturing agents produced distinct components which were identified in the ultracentrifuge. Combining this analysis with electron microscopy made it possible to identify the capsid and nucleocapsid, and a fragment having an s 20, w = 105 ± 10S. This fragment appeared spherical in the electron microscope and had dimensions between 12 nm and 16 nm. It was tentatively identified as the tail. The mol. wt. of the 105S particle and the empty head (200S) were calculated to be 2.3 × 106 and 21.7 × 106, respectively. Adding these weights to the estimated weight for DNA (25 × 106, Lang & Coates, 1968) gave a total weight for the phage of 49 × 106, equal to the mol. wt. obtained by Swaby (1959). In addition to these large fragments, two peptides were examined, one had a mol. wt. of 9300 ± 1400 in 6 m-GuHCl (1.5S) and appeared to be a single chain; the other, released when a suspension of phage was diluted, had a mol. wt. of less than 10000. The origins of the two peptides remain largely speculative, but in view of the marked associative properties of the 1.5S peptide and its detection only when the head was destroyed, it is likely that it was a binding fraction in the head of the phage. The second peptide may be more intimately associated with the DNA of T3.

Using a non-cytotoxic dose of ethidium bromide, a differential inhibitor of mitochondrial DNA and RNA synthesis, some early event(s), required after infection of Balb 3T3 cells with murine sarcoma virus, did not occur. This led to an inhibition of virus replication. The cells became refractory to inhibition 24 h after infection. The most effective inhibition was observed when cells were treated with ethidium bromide for 15 to 18 h before infection, suggesting the depletion of a cellular material involved in the establishment of virus infection. The degree of inhibition by ethidium bromide appeared to be affected by the multiplicity of infection.

Clonal subline of Kirsten murine sarcoma virus (Ki-MSV)-transformed non-producer BALB/3T3 (Ki-BALB) cells spontaneously produced flat variants with some properties of non-transformed cells at frequencies of 0.01 to 0.001. Such variants were epithelioid, contact-inhibited, grew to low saturation density and exhibited variable cloning efficiences in soft agar. They demonstrated no murine leukaemia group-specific antigen(s), no reverse transcriptase activity, and no infectious virus, but were agglutinable by concanavalin A. Murine leukaemia virus (MuLV) was induced after 5-iododeoxyuridine (IUDR) treatment from both the flat variants and from the parental Ki-BALB cells. However, Ki-MSV could not be rescued from these flat variants by superinfection with MuLV, nor induced by treatment with IUDR. The state of reversion was stable, revealing no back-transformation during 30 to 40 subcultures. The tumourigenicity of the flat variants in BALB/c mice was markedly reduced (actually undetectable) compared to that of the Ki-BALB cells. All flat variants were susceptible to MSV and MuLV infection. The loss of the transformed phenotype in spontaneous flat variants was associated with a decrease in chromosome number to a level similar to BALB/3T3 cells. These observations suggest that these revertants may have lost some or all of the Ki-MSV genome.

The replication of infectious LCM virus and its complement-fixing antigen was followed in cultivated foetal cells from colony-bred albino mice, foetal cells from CBA/Ca, C3H/HeJ, C57BL/6, and DBA/2 inbred mice, and L cells. Typical S-shaped growth curves were obtained in all instances and yields approached 104 intracerebral mouse ID50/cell. After the infection of newborn mice the multiplying virus rapidly reached a plateau of infectivity in tissues which remained thereafter. Ten weeks after neonatal infection the virus concentrations in blood and organs of ordinary albino mice and of CBA, C3H, C57BL/6, and DBA/2 inbred mice showed a wide range with highest levels in the kidneys and lowest in the blood.

The multiplication patterns and maximum yields of infectious virus and complement-fixing antigen were essentially identical in L cells and in foetal cells derived from five mouse strains; mutatis mutandis the same was true for persisting virus in the blood and organs of neonatal carrier mice.

The mechanisms involved in the interaction of complement components with sensitized equine arteritis virus (EAV) were investigated. Virus neutralization and virolysis depended on both the concentration of the complement components and the concentration of the sensitizing antibody. High concentrations of C4, 2 and 3, with an optimal concentration of C1, were sufficient for neutralizing virus infectivity in the presence of excess antibody. The addition of the remaining five components (C5 to C9) of the complement system induced lysis of the previously neutralized virus particle. Lysis was initiated by C8 and was augmented by C9. Components C5 to C9 did not enhance neutralization produced by excess antibody and limiting concentrations of complement components. In contrast, addition of components C5 to C9 enhanced neutralization by means of lysis of the virus particle under conditions of low antibody concentration.

IBT-related compounds were examined for their ability to inhibit wild-type and IBT-resistant mutants of vaccinia virus and for their capability to support the growth of an IBT-dependent mutant. Among thirteen compounds tested, six did not affect any one of the three virus strains and five behaved similarly to IBT, but two inhibited the growth of all three viruses and therefore differed from IBT. It was found that the last two compounds also differed from IBT in that they interfered with synthesis of the virus DNA. This difference is surprising in view of a substantial similarity in chemical structure.

The susceptibility of tobacco protoplasts to infection with cowpea chlorotic mottle virus varied with the age both of the plants and leaves used as a source for the protoplasts. The efficiency of infection for freshly prepared and stored protoplasts could be increased by an improved direct inoculation procedure. Poly-l-ornithine, poly-l-arginine, poly-l-lysine and poly-d-lysine were all effective in inducing infection, the first stage of which may occur within 20 s of exposure to inoculum. The effects of temperature, injury and sodium azide on infection suggested that the limiting process during inoculation is not controlled by enzymes. The yield of virus per infected protoplast was decreased by some antibiotics but not affected by various other additives to the culture medium.

The RNA of broad bean mottle virus (BBMV), cowpea chlorotic mottle virus (CCMV), and cucumber mosaic virus (CMV) bound tyrosine, as does brome mosaic virus (BMV) RNA. Other amino acids were not bound. Comparison of the efficiency of synthetase enzymes from Escherichia coli, bean, wheat, and Saccharomyces cerevisiae in aminoacylation of RNA from these viruses, and from tobacco mosaic virus (TMV) and turnip yellow mosaic virus (TYMV), showed that the plant enzymes were effective in tyrosylation of RNA from bromoviruses and CMV, whereas the yeast and bacterial enzymes were ineffective. The E. coli enzyme did not esterify TMV RNA with histidine, and the bean enzyme was poor in this ability. All enzymes were able to catalyse valine binding to TYMV RNA. However, 40 mm-KCl inhibited valine binding to TYMV RNA by the bacterial enzyme while its ability to catalyse binding by tRNA was not affected.

Using indirect immunofluorescence and in situ hybridization techniques the synthesis of structural proteins (hexon and fibre), T-antigen and virus DNA were studied in infected rat embryo cells early after infection by adenovirus type 2. The data obtained from this study, including infectious virus yields, indicate that this is a semi-permissive system in which transformation occurs. The adenovirus type 2/rat embryo transformation system was found to be reproducible and the transformation frequency was influenced by the tissue of origin and not by the strain of rat. A comparison of the in vitro characteristics of cells transformed by either adenovirus type 2 (Ad-2) or type 12 (Ad-12) is reported. No obvious property was observed which would explain the different in vivo behaviour of rat cells transformed by these adenovirus serotypes.

The structural proteins of 14 strains of Newcastle disease virus (NDV) were examined on reduced polyacrylamide gels. Three major and seven minor virus proteins (VP) were found reproducibly, one of the major and one of the minor poly-peptides being glycoproteins (VGP). The three major polypeptides had mol. wt. of 75000 (VGP75), 55000 (VP55) and 42000 (VP42) and the minor polypeptides 180000 (VP180), 110000 (VP110), 55000 (VGP55), 53000 (VP53), 52000 (VP52), 51000 (VP51) and 49000 (VP49). On polyacrylamide gel electrophoresis in a non-reduced system one of the minor components, VGP55, migrated to an apparently higher mol. wt. position (between VGP75 and VP55) with all strains of virus examined. Under the same conditions, it was found that VGP75 from some strains was either absent or present in greatly diminished amounts, and a new high mol. wt. glycoprotein appeared. By extraction of this high mol. wt. protein from non-reduced polyacrylamide gels, and electrophoresis of the reduced protein under reduced conditions the major component was found to be VGP75, but VP55 was also present. Amino acid analysis of the three major proteins from three strains of virus showed clear differences between the proteins of the different strains.

The proteins of purified polyhedra of the cytoplasmic polyhedrosis virus (CPV) of Nymphalis io were examined by electrophoresis in polyacrylamide gels containing SDS. The major polypeptide in polyhedra, and in inclusion body protein (poly-hedral protein) had a mol. wt. of 37000, and stained positively for carbohydrate. Purified virus particles contained three polypeptides, with mol. wt. of 116000, 109000 and 30000. RNA extracted from the virus particles had a melting profile characteristic of double-stranded RNA. Nine bands were resolved when this RNA was electrophoresed through 3% polyacrylamide gels. A comparison of the molar proportions of these segments suggests that there are 10 pieces of RNA, which form a genome with a mol. wt. of 14.4 × 106. There was good agreement between the sizes of the structural polypeptides, and the estimated coding capacity of four of the RNA segments. CPV virus particles share features in common with reovirus and, in particular, with the ‘core’ particles obtained by the enzymic digestion of intact reovirus particles.

Potato virus X reacted with reagents commonly used for protein amino groups, and some of its properties were changed. 2,4,6-trinitrobenzenesulphonic acid, pyridoxal-5-phosphate and methyl picolinimidate altered its absorption spectrum; the last two altered its fluorescence spectrum, and the first two altered its electrophoretic mobility. These reagents did not necessarily inactivate the virus; preparations judged to contain two modified amino groups per protein subunit retained 50 to 100% of their initial infectivity. This supports the previous conclusion that PVX-Q, an infective product of PVX and an oxidized leaf phenol, contains modified lysine ε-amino groups.

The c.p.e. of herpes simplex virus was studied by scanning electron microscopy. The infected cells showed changes in size, shape, numbers of microvilli, numbers and integrity of intercellular bridges, and surface of the monolayer. When substantiated, these alterations may prove to represent the early phases of cellular reaction to virus invasion.

DNA extracted from a chicken lymphoblastoid cell line (MSB-1) originally derived from a Marek’s disease tumour was examined for the presence of the Marek’s disease virus genome. DNA cRNA membrane filter hybridization experiments established the presence of 60 to 90 genome equivalents in these cells.

In tobacco (Nicotiana tabacum cv. Xanthi-nc) actinomycin D inhibits the resistance to tobacco mosaic virus that is induced by polyacrylic acid or by earlier infection with potato virus Y. Formation of the additional proteins associated with this resistance is also prevented.