- Volume 11, Issue 3, 1971

A large icosahedral bacteriophage, Si1, infectious for Spirillum itersonii has been isolated and characterized. It contains double-stranded DNA which has 53% G+C, no unusual bases and a molecular weight 2·6 x 107.

Purified poliovirus preparations were heated and analysed by sucrose gradient centrifugation. They consisted of virus-like particles containing RNA and sedimenting at about 80s, empty 80s capsids, 35s virus RNA, and a non-sedimentable capsid polypeptide (VP 4). By electron microscopy the 80s ribonucleoprotein particles (80s RNP) were similar in appearance to intact poliovirus particles. They contained infectious, RNase-sensitive RNA that could be liberated from the capsid by treatment at room temperature with 1% sodium dodecyl sulphate. One of the virus polypeptides was missing, and they had lost the antigenicity of the mature virus and the ability to adsorb to HeLa cells.

Degradation of poliovirus particles probably occurs in two steps: the first is the splitting off of a minor part of the capsid protein (VP 4) followed under certain conditions by a liberation of the RNA from the capsid. The alteration of the physical and biological properties of the virus particle is probably due to the loss of the protein rather than to the liberation of the RNA.

The multiplication of four strains of tobacco mosaic virus was compared at temperatures of 20° to 25° and at 35° by estimating the concentration of total infective virus RNA, intact virus, virus antigen and insoluble virus protein in plants at different times after inoculation. The four strains were: the type strain, nitrous acid mutants pm2 and ni118, and the ‘thermophilic’ strain tc. The concentration of total infective RNA of all four strains reached its maximum concentration about a week after inoculation and was about ten times greater at 20° than at 35°, but the infectivity of intact virus and the virus antigen titre varied with the strain. The intact virus and virus antigen concentrations of the type strain were reduced similarly to the RNA concentration when the temperature was raised. Ni118 produced very little infective intact virus at 35° but as much at 20° as the type strain, although the particles were less well-formed. No intact virus of pm2 was found at any temperature as the virus protein is non-functional. The strain tc produced about as much infective intact virus as 35° as the type strain at that temperature but much less virus at 20° than at 35°.

Virus multiplication of the four strains was affected by increased temperature in two ways: (1) the replication of the RNA of all strains was inhibited; (2) the RNA of those strains which had defective protein was degraded. Degradation was most obvious with pm2, the infectivity of which, after a maximum was reached, declined at increasing rates with increasing temperature. Free RNA of ni118 accumulated at 35° because the protein became insoluble but, in contrast to pm2, some complete virus was produced. There was no free RNA in plants infected with type strain. The apparently greater infectivity of tc at 35° than at 20° resulted from the fact that its RNA is badly coated at 20°, forming unstable particles. The concentrations of total infective RNA of type strain and tc at 35° did not differ.

When infected plants were transferred from 35° to 20°, the infectivity of intact virus increased with the type strain and still more with Ni118.

Strain ni 118 of tobacco mosaic virus in tobacco plants kept at 35° exists mainly as free virus RNA and insoluble virus protein but also forms a few intact virus particles. Buffer extracts of infected leaves have, therefore, very little infectivity. Similar extracts from plants inoculated with a mixture of ni 118 and the type strain tested on plants in which only ni 118 gives symptoms are very much more infective (40 to 500 times). The increase in the ni 118 infectivity of leaf extracts is even greater when the leaves are infected with the Nigerian cowpea virus instead of the type strain. The cowpea virus is a strain of tobacco mosaic virus only slightly serologically related to ni 118. Neutralization of infectivity tests, using homologous and heterologous antisera, showed that the increased infectivity of ni 118 in dual infections is caused by the nucleic acid of ni 118 being incorporated in protein of Nigerian cowpea virus.

The molecular size of ‘early’ and ‘late’ SV 40-specific RNA synthesized in productively infected monkey kidney cells was investigated. Labelled RNA extracted from infected cells was fractionated by velocity centrifugation in a sucrose density gradient and then hybridized with excess, unlabelled SV40 DNA. The largest component in preparations of ‘early’ SV 40-specific RNA, which was synthesized in actinomycin D-treated cells, had a molecular weight of approximately 1·2 × 106. Late after infection, the molecular weight of the predominant species of cytoplasmic SV 40-specific RNA was about 1·7 × 106. The latter molecular size corresponds to the full transcription of one SV 40 DNA strand into a polycistronic messenger RNA. The major portion of nuclear ‘late’ virusspecific RNA sedimented at 32 to 50s (molecular weight range: 2·3 to 5·7 × 106) , indicating that in the nuclei of productively infected cells the virus genome can be transcribed into RNA molecules much larger than a single virus DNA strand.

The mutant Ni 118 obtained from tobacco mosaic virus (TMV) treated with nitrous acid produces defective coat protein, especially when multiplying in plants kept at 35°. At this temperature, few complete virus particles are formed, and extracts of infected plants contain mostly free infective RNA and insoluble coat protein. At 20°, however, Ni 118 multiplies as well as the type strain of TMV ( Jockusch, 1966 ; Kassanis & Bastow, 1971a ). To gain more information about the conditions in infected cells we examined them by light and electron microscopy. Light microscopy was done by phase-contrast on living cells from epidermal strips of the undersides of Samsun tobacco leaves 1 week after inoculation. For electron microscopy the methods of Milne (1970) were used; pieces of leaf were fixed in glutaraldehyde and then osmium tetroxide, dehydrated in acetone, soaked in uranyl acetate in acetone and embedded in Epon. After sectioning, the material was stained in lead citrate.