- Volume 39, Issue 3, 1978

Vaccinia virus-induced morphological lesions were studied in LLC-MK2, HeLa and L cells. In LLC-MK2 cells, cell rounding occurs within 30 to 60 min after infection with 300, 900 or 2700 particles/cell and the time of appearance of these changes is dependent on the multiplicity of infection (m.o.i.). When cycloheximide (300 µg/ml) is added to cultures at the time of infection, early cell rounding is prevented regardless of the m.o.i. However, cell rounding does occur when cycloheximide is removed, and its time of appearance and extent depends upon the time of removal of the compound and the m.o.i. Upon removal of cycloheximide at 1 or 2 h after infection early cell rounding occurs, and virus polypeptide synthesis is evident in cells infected at all three multiplicities. However, when the drug is removed at 4 h after infection, cell rounding and virus polypeptide synthesis occur only in cultures infected at 300 particles/cell. Early morphological changes are also prevented in HeLa and L cells infected at 300 particles/cell in the presence of cycloheximide. These changes occur only if the compound is removed up to 2 h after infection in HeLa cells and up to 40 min after infection in L cells. Early morphological lesions are not seen if the compound is removed at later times. The occurrence of early morphological changes in HeLa and L cells is also correlated with the synthesis of virus polypeptides. All cell types, when infected at 2700 particles/cell in the presence of cycloheximide, or inhibitors of RNA synthesis, display cell fusion. Thus, whereas early morphological changes require virus protein synthesis to become manifest, cell fusion occurs in the absence of virus RNA or protein synthesis and may be mediated by a component of the virion.

Heat-inactivated murine cytomegalovirus (MCMV) stimulates cellular DNA synthesis in WME, NMG, 3T3, Wg1A, chick and NIK-8 cells, but active or u.v.-irradiated MCMV does not. The stimulation of DNA synthesis in NIL-8 and chick cells was studied in detail. We found that both the nuclear and the mitochondrial DNA synthesis were stimulated in these cells. There was no virus DNA synthesis during the period we studied (48 h). The stimulation of nuclear DNA synthesis was about threefold in NIL-8 and 2.5-fold in chick cells as measured by the rate of incorporation of 3H-thymidine (3H-dThd) in the CsCl fractions which banded at the density of cell DNA. The stimulation was about 9.5-fold in NIL-8 and 1.7-fold in chick cells as detected by autoradiography. There was a 3-fold and a 2.2-fold increase in the degree of incorporation of 3H-dThd into mitochondrial DNA of NIL-8 and chick cells, respectively. The amount of mitochondrial DNA obtained in infected cells of both kinds was about twice that in control cells. The synthesis of mitochondrial DNA was also stimulated by a factor of 2 in the thymidine kinaseless 3T3 cells which incorporate exogenous thymidine exclusively into mitochondrial DNA. There were no MCMV specific antigens detectable by immunofluorescence 5 h after infection, but diffuse nuclear fluorescence could be demonstrated 24 h after infection. Our results indicate that the heat-inactivated virus penetrates the cells, stimulates host DNA synthesis and induces synthesis of early MCMV antigens.

Spontaneous production of endogenous xenotropic viruses by clones of New Zealand Black (NZB) embryo cells occurs at a constant level even after several cell transfers. Foci of cell alteration occur spontaneously in some of the clonal lines after 6 to 10 passages. The amount of virus generated does not correlate with spontaneous transformation of these NZB cells nor of cells from NIH Swiss or BALB/c mice. The data demonstrate an intracellular regulation of xenotropic virus expression which remains stable over several cell generations and differs from that controlling cell transformation.

Reovirus infection inhibits the incorporation of 3H-thymidine into cellular DNA. We have now investigated several aspects of this inhibition in L-929 cells early (8 h) after infection at high multiplicity (200 to 250 p.f.u./cell). Using equilibrium sedimentation analysis of DNA sequentially labelled with density and radioactive analogues of thymidine, we find a 52% reduction in the amount of DNA synthesized with no change in rate of replication fork movement in infected cells. Gel electrophoresis of histones labelled with 3H-lysine shows that infection inhibits their synthesis by 76% several hours before overall cellular protein synthesis is inhibited. There is also a reduction of nearly 50% in the size of the thymidine triphosphate pool, as measured by enzymic assay. The proportion of exogenous nucleotide in the pool is the same as in uninfected cells since there is no change in the buoyant density of DNA labelled during a short pulse with 3H-bromodeoxyuridine. The uptake of thymidine is reduced, but its phosphorylation to thymidine triphosphate is normal. The findings provide direct evidence that DNA synthesis is inhibited early in infection. This inhibition is accompanied by other derangements of thymidine and chromatin metabolism suggesting that there is an early and specific attack by reovirus on nuclear function in infected cells.

Changes induced in the ionic composition of the surface of herpes simplex virus (HSV) infected cells were evaluated by whole cell electrophoresis and isoelectric focusing. The kinetics of the progressive decline in cell electrophoretic mobility (a measurement of negative surface charge density) after infection corresponded to the production of infectious virus. The observed HSV-induced increase in cell isoelectric focusing pH demonstrated that the electrokinetic cell surface became ionically more positive. The nature of the ionic changes were investigated using a series of specific reagents in conjunction with cell electrophoresis. Infected cells had 2.4 times more surface binding sites for the amino-specific reagent 4-acetamide-4′-isothiocyanostilbene-2,2′-disulphonic acid. A relationship between these virus-induced amino groups and HSV surface antigens was indicated by the similar kinetics of their appearance and electrophoretic neutralization by HSV antibodies.

When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 °C) and shifted to the restrictive temperature (39 °C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al., 1977), and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 °C, indicating that no new active RNA polymerase is formed at 39 °C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 220000 (ns220) was demonstrated in ts-4 infected cultures shifted to 39 °C. Ns220 was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns220 are located near the initiation site at the 5′-end of the 42S RNA. The viral specificity of ns220 was shown by its disappearance after a shift to 28 °C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 °C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation.

The sizes of non-covalently linked RNA subunits isolated from highly leukaemogenic Friend virus derived from the plasma (PV, plasma virus) of leukaemic mice were compared to the RNA subunits isolated from low leukaemogenic Friend virus grown in tissue culture (TCV, tissue culture virus). Histograms derived from electron microscope measurements showed that about one-half of the plasma virus RNA was 1.4 to 2.5 µm in length, corresponding to a mol. wt. range from 1.8 × 106 to 3.2 × 106 and the other half less than 1.4 µm. In contrast, approx. 50% of the TCV RNA was only 0.7 to 1.6 µm in length (mol. wt. 0.9 × 106 to 2.0 × 106) and the remainder less than 0.7 µm in length regardless of whether the virus RNA was isolated from 3, 9, 36 or 72 h cultures. The histograms suggest size classes for both TCV and PV derived RNA subunits. Data obtained from sucrose gradient sedimentation of heat-denatured FLV RNA agreed with the electron microscope length measurements. The smaller sizes of the TCV RNA subunits are hypothetically related to the limited biological activity of Friend leukaemia virus produced from leukaemic cells in culture. Comparable results were obtained using two different RNA extraction procedures. Contamination of TCV nucleic acid preparations by cellular DNA was observed even when the virions were harvested from short term cultures and purified by isopycnic sucrose gradient centrifugation. Consequently, preparations of intact virus were treated with DNase prior to sucrose gradient purification of the virions.

A previous paper (Kimberlin & Walker, 1977) described an experimental model of scrapie in hamsters in which the incubation period decreased progressively over the first 4 passages before becoming stable at the 5th and subsequent passes. Studies have been made of some of the agent strains present in brains taken from the 2nd, 3rd, 4th and 6th hamster passes. The results indicate the presence of at least two strains of agent at the 3rd passage level. One of these (431K) is highly pathogenic for mice and the other (263K) has an extremely low pathogenicity for mice. However only one of these strains (263K) is present in hamster brain after the 6th serial passage. It is suggested that the ‘adaptation’ of scrapie to hamsters may involve the selection, from a mixture, of a single strain which is highly pathogenic for hamsters. The possibility of modification of the properties of agent strains on passage discussed.

The genomic RNA of human coronavirus strain 229E (HCV 229E) migrated on polyacrylamide gels as a single peak with a mol. wt. of 5.8 × 106. Denaturation of the genome with formaldehyde did not alter its electrophoretic mobility, which suggests that the HCV 229E genome is a single-stranded molecule. At least 30% of the genomic RNA was shown to contain covalently attached polyadenylic acid [poly(A)] sequences by binding the RNA to an oligo(dT)-cellulose column. These poly(A) tracts were shown to be about 70 nucleotides in length by measuring the resistance to digestion of HCV 229E RNA with pancreatic and T1 RNases. Finally, the genomic RNA was shown to terminate at or near the 3′-terminus on the basis of its susceptibility to polynucleotide phosphorylase.

Simple biochemical measurements have been shown to seriously overestimate the production of C-type particles by Chinese hamster ovary (CHO) cells treated with 5-bromodeoxyuridine. First, most particle-bound DNA polymerase activity released by induced cells was associated with particles which had a different density from C-type particles. Second, when labelled with radioactive uridine, induced CHO cells released small amounts of particle-bound radioactivity. Most of the radioactivity, however, was in DNA and did not sediment with the particle-bound polymerase. Thus, few particles which had RNA, an associated DNA polymerase, and the density typical of RNA tumour viruses were released by BrdUrd-induced CHO cells. In spite of this, some immature C-type forms were observed by electron microscopy in partially purified preparations of DNA polymerase-containing particles from induced CHO cells.

In cells infected with herpes simplex virus, HSV-1, newly synthesized polypeptides accumulated in the nucleus at different rates, which did not change during the first 6 h after infection. Canavanine, an arginine analogue, prevented the nuclear accumulation of ICP (infected cell polypeptides) 5 and 8, and azetidine, a proline analogue, prevented that of ICP 5 and 7. The transfer of polypeptides to the nucleus was inhibited at 4 °C but not by dinitrophenol. Some of the nuclear polypeptides could be released by washing isolated nuclei with hypertonic salt solutions. ICP 17 was particularly sensitive to high salt treatment while ICP 5 and 11 were resistant. ICP 4b, a modified form of the α polypeptide ICP 4, was released by EDTA, and the detergent NP40 removed ICP 11. Treatment of nuclei with DNase selectively reduced the amount of bound α polypeptides ICP 4c (the second modified form of ICP 4), 0 and 27 as well as ICP 8 and 25. Nuclei isolated from infected or uninfected cells and incubated in labelled cytoplasmic extracts took up primarily ICP 8 and 32. Alpha polypeptides were takzen up to a lesser extent and ICP 6 and 10 were excluded. It is concluded that affinities for various constituents of host cell nuclei are likely to determine the nuclear accumulation of specific virus polypeptides.

High mol. wt. DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious. Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E. coli (lambda c857 tsxisam6 ) lysogen yielded 200 times as many plaques on sup + than on sup − spheroplasts. Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not. About 104 genome equivalents of E. coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments.

Isolates of feline calicivirus (FCV) can be divided into four groups according to plaque size under an agar overlay. All isolates classified as minute plaque, along with certain other isolates, are sensitive to inhibitors present in agar.

A procedure is described by which human leukocyte interferon can be concentrated 5000-fold and purified over 100-fold on a large scale with over 50% recovery.

The structure of the ribonucleoprotein (RNP) complex of three coronaviruses was investigated. A single-stranded helix of diam. 14 to 16 nm and up to 320 nm in length was released from disrupted particles of human coronavirus strain 229E and mouse hepatitis virus strain 3 after incubation in mild conditions. The helical complexes appeared to be composed of globular subunits with long axes of 5 to 7 nm surrounding a hollow core of diam. 3 to 4 nm. The complexes were shown to be sensitive to both pancreatic RNase and to pronase. No undegraded internal component was obtained from disrupted avian infectious bronchitis virus particles. We conclude that these structures are RNP complexes. The similarity between these RNPs and those of other large lipid containing RNA viruses is discussed.

Intracellular K+ ion concentration and transport were measured in L cells infected with wild type VSV, which rapidly inhibits total protein synthesis in infected cells, and with a mutant, R1, defective in this function. No alterations in intracellular K+ ion concentration or transport were observed until late in infection and the late changes seen were similar for both viruses. Thus the inhibition of total protein synthesis by VSV cannot be ascribed to virus induced changes in host K+ ion concentration.

Four of eight mycobacteriophages did not form plaques after they were exposed to chloroform. Phages sensitive to chloroform did not produce plaques when plated on media containing 1000 µg/ml of streptomycin sulphate. The same concentration of dihydrostreptomycin sulphate did not interfere with plaque formation. Mutants of Mycobacterium smegmatis resistant to each of the eight phages were isolated. Sensitivity or resistance to chloroform and streptomycin sulphate and phage resistant bacterial mutants may provide a basis for classifying the mycobacteriophages.

The RNA of the sinuous papaya mosaic virus particles was labelled in vivo with 32P or in vitro after oxidation by periodate and reduction by KB3H4. After digestion with RNases T1, T2 and A, the oligonucleotides were fractionated by electrophoresis or by chromatography on a DEAE-cellulose column. After such fractionation, an oligonucleotide resistant to the RNases and phosphatase with a structure of m7G5′ppp5′Gp was obtained from the 5′-terminus of the RNA.

Lymphocytic choriomeningitis virus strain WE multiplied in cultivated peritoneal macrophages from unstimulated NMRI mice. As revealed by immunofluorescence technique, most cells participated in the infectious process. They were not, however, functionally altered. Morphologically, infected cells contained more cytoplasmic vacuoles, but these were also present, though less numerous, in uninfected ageing cells and in cells having phagocytosed Latex beads. As compared with the WE strain virus, the E-350 (Armstrong) strain multiplied more slowly to lower titres, but the proportion of infected cells was similar.