- Volume 21, Issue 3, 1973

By use of [125I]-p-iodophenylisothiocyanate in a modified Edman procedure, N-terminal aspartate, glycine and serine were identified and approximately assayed in the capsid protein of purified poliovirus, strain Mahoney. Two separate preparations of virus gave similar results. These three N-terminal amino acids were recovered in amounts consistent with about 60 molecules of each per virus particle, or 1 mol of each per mol of virus protein.

Denatured DNA of herpes simplex virus was released from the particles using an alkaline detergent, Decon-75. The largest single strands sedimented on alkaline sucrose gradients with a mol. wt. of 47.2 ± 0.33 × 106, slightly less than half the value calculated for the intact duplex (104 × 106). About 50% of the DNA was found in fragments which sedimented slower than this in a heterogeneous manner. On agarose gel electrophoresis the largest strands migrated with a mol. wt. of 40 × 106. The reason for this difference is not known but since the individual strands of T 4 DNA were shown to migrate with slightly different R f values, factors other than mol. wt. may affect the migration of single-stranded DNA. Two fragments of mol. wt. 35 × 106 and 30 × 106 were observed but the rest of the fragments remained unresolved by this technique. Virus DNA associated with the nucleus of infected cells had a much lower mol. wt. than particle DNA (3.2 × 106). Although this value increased if the DNA was isolated from intact cells, the average sedimentation coefficient of nuclear virus DNA was never as high as the largest strands of particle DNA.

When the largest single strands from particles were prepared by sucrose gradient fractionation they exhibited a unimodal mol. wt. distribution after both sedimentation and electrophoretic analysis. These ‘intact’ single strands were annealed and analysed by banding in CsCl gradients and by analyses employing the Neurospora crassa endonuclease. The results show that the ‘intact’ strands reassociated with the same kinetics and to the same extent as total virus DNA, suggesting that both strands of the duplex were present in equal amounts.

The characteristics of IBT-dependent (IBT d ) and IBT-resistant (IBT r ) mutants of vaccinia virus were compared with those of the wild type (wt) strain. The mutants did not differ from the wild type strain by their sedimentation in sucrose gradients. Minor differences in the polypeptide composition of the virus particles and in the neutralization of the three virus strains by anti-vaccinia human immunoglobulin were observed. Mixed infections of the viruses in HeLa cells enabled the growth of the strains under their unfavourable conditions (wt in the presence of IBT and IBT d in the absence of IBT).

The IBT-dependent mutant of vaccinia virus needs IBT for its growth. IBT is not essential for the synthesis of virus DNA, but the formed DNA does not become resistant to deoxyribonuclease in its absence. Both ‘early’ and ‘late’ virus proteins are synthesized in cells infected with the mutant, but in the absence of IBT one of the virus core structural polypeptides, which is normally formed from a higher mol. wt. precursor, is not made. These findings suggest that the IBT-dependent step of the mutant occurs earlier in the maturation process of the virus than does the block produced by IBT with the wild-type strain.

Infectious pancreatic necrosis (IPN) virus has been purified and some physicochemical properties of its nucleic acid and protein moieties have been determined. The buoyant density of the virus particle in CsCl is 1.35 g/ml; degraded particle banded at a density of 1.31 g/ml. The virus nucleic acid was found to be double-stranded, according to several different criteria, namely resistance to pancreatic RNase, low density in Cs2SO4 (1.615), sharp melting curve with a Tm of 89 °C in hypotonic buffer, and base composition. In a sucrose gradient the bulk of the virus RNA banded as a single peak (14 S); a small part of the virus nucleic acid was found at the top of the gradient. By gel electrophoresis virus RNA was resolved into three peaks corresponding to double-stranded molecules with mol. wt. ranging from 2.85 × 106 to 2.55 × 106. Gel analysis of virus proteins revealed three polypeptides of mol. wt. 80, 50 and 30 × 103, representing respectively 3%, 68% and 29% of the total protein. On the basis of these results IPN virus appears to be related to some reo-like viruses, having a single protein shell and a single class of double-stranded RNA.

Some properties of two temperature-sensitive mutants of tobacco rattle virus are described. Experiments with mixtures of long and short particles from the mutants and the wild-type (CAM) strain showed that both mutations are in the RNA of the long particle. The mutants could not be distinguished from each other or from the wild-type virus by heat inactivation, serology, particle length or symptoms produced in a range of host plants under normal glasshouse conditions. One of the mutants was temperature-sensitive in local lesion formation in Chenopodium amaranticolor, and in production of infectious virus and RNA in Nicotiana clevelandii, whereas the other appeared to be temperature-sensitive only in C. amaranticolor. Some possible reasons for this behaviour were investigated.

The properties of a mutant of cowpea chlorotic mottle virus which, although infective, cannot be disassembled in 1 m-NaCl, pH 7.5, are described. The mutant coat protein contains a lysyl to arginyl replacement which affects the hydrodynamic and titration characteristics of the virus which are discussed in regard to the anomalous ionization of a carboxyl-carboxylate pair and a lysyl residue.

Measurement of replicating molecules of polyoma virus DNA after digestion with endonuclease R1 shows that DNA replication is bidirectional, starting predominantly at a specific site. In both large plaque and small plaque polyoma virus DNA this site is 29 ± 2% of the total length of the DNA from the cleavage site of the endonuclease R1.

RD-114 virus rapidly induces fusion of the KC cell line, a human malignant glioma cell transformed by Rous sarcoma virus. Treatment of the virus with trypsin, heat, ultrasonic vibration, or ether, completely eliminated fusion activity, while deoxyribonuclease (DNase), ribonuclease (RNase), or neuraminidase treatment had no effect. β-Propiolactone inactivation of the virus resulted in no decrease of fusion activity, though infectivity was completely lost. The KC cell line was productively infected with RD-114 virus, and in this condition was completely refractory to fusion by RD-114, either in response to endogenous virus or to large amounts of exogenous virus. Treatment of the normal KC cells with actinomycin C, cytosine β-d-arabinofuranoside, or cycloheximide did not prevent fusion in response to RD-114 virus. These data suggest that intact, but not necessarily infectious, virus is required for fusion, and that the process is very similar to the fusion of XC cells by murine leukaemia viruses.

Electron microscopy of L cells infected with vaccinia virus at a high multiplicity indicated that virus cores entered the cytoplasm, during a 15 min period of adsorption at 37 °C, as a result of fusion between the plasma membrane and the envelopes of virus particles. It is suggested that for vertebrate poxviruses this, rather than phagocytosis, could be the effective mode of entry leading to infection.

The properties of a new brucella phage isolated from culture fluids of Brucella suis and designated the Weybridge phage, were examined. The phage was lytic for smooth Br. abortus, Br. suis and Br. neotomae cultures but there was no lysis of Br. melitensis, Br. canis or Br. ovis strains. The host range, adsorption pattern, chemical stability and particle morphology were similar to those reported for brucella phages M51 and S708. However, phage neutralization tests established that Weybridge phage was serologically distinct from M51 and S708 although the three phages possessed antigenic determinants in common with each other and with the Tbilisi reference phage. One-step growth experiments of Weybridge phage on Br. abortus, Br. suis and Br. neotomae are described. No evidence of lysogeny was found although the origins of the phage were suggestive of this.