- Volume 19, Issue 1, 1973

The production of interferon by human diploid cells stimulated by poly-I:C can be increased by pretreatment of the cells with interferon (priming effect). Large amounts of interferon (30000 units/ml) can be obtained by combining priming with a superinduction schedule adapted from that described for rabbit kidney cultures (Tan et al., 1970; Vilcek & Ng, 1971). Human plasma protein could replace bovine serum albumin in the production medium.

Dithiothreitol (DTT), a reagent which cleaves disulphide bonds, destroyed the haemagglutinin, neuraminidase and ‘haemolysin’ of Sendai virus. The treated virus particles appeared morphologically intact. They failed to bind haemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibodies. Antisera against DTT-treated virus inhibited haemolysis induced by intact virus, although they did not contain detectable HI, NI or virus-neutralizing antibodies. Treated and untreated virus particles were labelled with [14C]-iodoacetamide or with [14C]-N-ethylmaleimide, disrupted with SDS and subsequently electrophoresed in polyacrylamide gels. Thus, polypeptide species with accessible cysteinyl residues, either originally present in intact virus or resulting from reduction of disulphide bonds, were distinguished. The significance of these findings is discussed.

Treatment of Sendai virus with 0.3% tri(n-butyl)phosphate and 0.1% Tween 80 led to the disintegration of virus envelopes and to the formation of envelope-derived particles (EP) consisting of ‘rosettes’ endowed with surface projections and of spikeless envelope remnants. EP had a higher buoyant density in CsCl gradients than intact virus (1.29 compared with 1.20 g/cm3); their sedimentation coefficient was 190 S. EP agglutinated red blood cells and contained neuraminidase. Their haemolytic activity was greatly increased in comparison with intact virus. Three virus polypeptides and traces of an additional protein were detected in EP by polyacrylamide gel electrophoresis. EP retained approximately 30% of choline-containing phospholipids present in the virus envelope. The haemolytic activity of EP and of intact virus was destroyed by treatment with either trypsin or diisopropylfluorophosphate. Insolubilised trypsin failed to alter the haemolytic activity. Proteolytic cleavage of RBC membrane components occurred during haemolysis.

Tanapox and Yaba viruses have the same host range and are similar in size, shape and ultrastructure. However, the lesions produced by the two viruses differ markedly both in gross and histological appearance. Yaba lesions in either monkey or man are proliferative and involve mesodermal cells while Tanapox virus affects almost exclusively the epidermis. In tissue culture both viruses grow only in cells of human or primate origin, but while Yaba virus in BSC-1 cells produces small heaped up tumour-like masses, Tanapox virus induces intense granularity and degeneration of the infected cells. Nuclear vacuolation which is a feature of Tanapox infection has not been observed in Yaba lesions.

Monkeys recovered from infection with either virus show partial immunity to the other; further immunizing injections of virus may lead to complete resistance to challenge with large doses of the heterologous virus. Serological comparison of the viruses by complement fixation, precipitation and neutralization tests using antisera prepared in monkeys shows some degree of cross reactivity. Tests with absorbed immune sera suggest that each virus possesses, in addition to a common antigen, an antigen specific to itself; antibody to a specific antigen can also be demonstrated by neutralization tests.

A collection of temperature-sensitive mutants of influenza virus was examined in an attempt to define their defective functions when grown at the restrictive temperature and to detect alterations in the physical properties of virus grown at the permissive temperature. Three mutants were found to be defective in the synthesis of haemagglutinin. The haemagglutinin of the fourth mutant was shown to be functionally and antigenically normal but it could not be detected at the cell surface. A similar absence of function at the cell surface was demonstrated for the neuraminidase of two other mutants. Two genetically identical haemagglutinin-defective mutants were also partially defective in the incorporation of uridine into virus RNA but this pleiotropy did not extend to the neuraminidase. Although immunofluorescence showed that all mutants were able to synthesize ribonucleoprotein, the ribonucleoprotein of one mutant was unable to migrate from the nucleus. The heat-sensitivity of one mutant could not be correlated with defective function.

The physical properties of mutant virus particles did not differ from the parent strain except that the infectivity of one mutant was more labile and another mutant had an altered affinity for erythrocytes of different species. These defects did not correlate with known defects in multiplication.

Vaccinia virus-induced antigens and polypeptides labelled late in infection with [14C]-leucine were examined in gel precipitin tests and polyacrylamide gel electrophoresis followed by autoradiography. Pulse-chase experiments, investigations on the effects of rifampicin and direct analysis of precipitin lines by gel electrophoresis allowed the identification of two major precipitin lines in terms of the polypeptides specific for the infected cell. One of these was non-structural. A small proportion of the other antigen was incorporated into sedimentable structures during a chase; this was prevented by rifampicin. Evidence suggesting that three virus-specified polypeptides are formed by cleavage of larger precursors is also presented and discussed.

Treatment of mouse L 929 cells in cell culture with mouse interferon, prior to infection with reovirus type 3, decreased the amount of virus progeny formed. In infected, interferon-treated cells less reovirus specified double-stranded RNA (ds reoRNA) and single-stranded RNA accumulated than in infected control cells and the rate of ds reoRNA accumulation was inversely related to the concentration of interferon with which the cells were treated prior to infection. Since ds reoRNA can be conveniently assayed, this forms the basis of a fast and simple assay for interferons.

Purified Semliki Forest virus was analysed by analytical sedimentation, rate zonal sedimentation, isopycnic sedimentation, polyacrylamide gel electrophoresis and electron microscopy. It was found that the spherical virus particles had a sedimentation coefficient of 241 S (249 S determined by rate zonal sedimentation), and a buoyant density in sucrose of 1.190 g/ml; one protein and one glycoprotein were present in the virus; after fixation and negative staining with phosphotungstic acid the diameter of the particles was measured to be 50 to 56 nm. Treatment of Semliki Forest virus with 0.1% Tween 80 and 1% tri(n-butyl)phosphate (TNBP) for 1 h at 4 °C destroyed infectivity and resulted in the appearance of the following haemagglutinating and immunizing virus components. (1) A spherical 211 S (217 S) component, 69 to 75 nm in diameter, consisting of an envelope and a core; one protein and one glycoprotein were present; the [3H]-uridine:[14C)-protein ratio was 2.8-fold higher than that of double-labelled untreated virus. (2) A spherical component, 70 to 73 nm in diameter, consisting of the virus envelope lacking intact cores; s 20,w = 140 S (153 S); one glycoprotein was detected; the [3H]-uridine: [14C]-protein ratio was about 1/3 of untreated virus. (3) A 135 S component. (4) Structures assumed to represent envelope fragments; much of them had a sedimentation coefficient of 49 S. It was concluded from experiments with [3H]-uridine-[14C]-amino acids double-labelled and [14C]-stearic acid-labelled virus that Tween 80-TNBP dissociates lipids and some protein components from the virus.

Parental and clonal lines of SV40-transformed human embryonic lung (WI38 Va13A) cells spontaneously produce infectious virus which is temperature-resistant (tr), i.e. replicates in monkey kidney cells at either 38.7 °C or 33.5 °C. Superinfection of WI38 Va13A cells lines with DNA from a temperature-sensitive (ts) SV40 mutant at the permissive temperature (33.5 °C) did not enhance the yield of tr SV40 as would be expected if the superinfecting ts SV40 DNA neutralized an SV40-specific repressor, or if the ts SV40 DNA supplied a virus-specific excision function and/or post-excision functions for the maturation of the resident tr SV40. The failure of superinfecting ts SV40 to enhance replication of the resident tr SV40 of WI38 Va13A cells is not due merely to a competitive advantage of the replicating superinfecting SV40 over the endogenous SV40. Experiments were carried out with 1-β-d-arabinofuranosylcytosine (ara-C) treated and ts SV40 DNA-infected WI38 Va13A cells to permit expression of early ts SV40 functions and to synchronize replication of ts SV40 DNA and the newly excised endogenous SV40 DNA. Ara-C treatment did not enhance formation of the endogenous tr virus. In further competition experiments, CV-1 cells were preinfected with ts SV40 particles and superinfected 6 to 24 h later with tr SV40 DNA recovered from SV40 spontaneously produced by WI38 Va13A cells. Even when the interval between the first and second infection was 24 h, very significant amounts of tr SV40 synthesis occurred and 28% of doubly infected cells still produced temperature-resistant infectious centres. The experiments suggest that rescue of the resident SV40 is determined primarily by cellular rather than superinfecting SV40 gene functions.

Interaction of [3H]-labelled poly(rI).poly(rC) with the cell has been studied in several cell cultures which differ markedly in their sensitivity to the antiviral activity of the polynucleotide (in order of decreasing sensitivity): primary rabbit kidney (PRK) cells, human skin fibroblasts (HSF), mouse embryo fibroblasts (MEF), mouse L-929 cells, rabbit kidney (RK 13) cells, HeLa, BSC-1 and VERO cells.

No significant differences were noted in the amounts of either total or acid-insoluble radioactivity associated with the cell at various times, following exposure of [3H]-poly(rI).poly(rC) to these different cell cultures. No significant differences were noted in the fate of cell-bound [3H]-poly(rI).poly(rC) in four out of the eight cell cultures tested. However, significant differences were observed in the sensitivity of cell-bound [3H]-poly(rI).poly(rC) to extraneous ribonuclease treatment: [3H]-poly(rI).poly(rC) bound to the cells that are most sensitive to the antiviral activity of the polynucleotide (PRK, HSF, MEF, L-929, RK 13) appeared to be markedly more accessible to ribonuclease treatment than [3H]-poly(rI).poly(rC) bound to cells that are rather insensitive to the antiviral activity of the polynucleotide (HeLa, BSC-1, VERO).