- Volume 29, Issue 1, 1975

N-tropic Friend leukaemia virus (FLV) was serially passaged in a B-type C57BL/6 mouse cell line, YH-7. After a single passage, the infectious efficiency of FLV in the restrictive YH-7 cells was significantly increased. This adapted character of FLV could be reversed by a single passage in permissive N-type MLg cells. The true host range conversion from N to NB was accomplished after eleven to twelve passages in YH-7 cells. In contrast, the host range conversion of N-tropic murine sarcoma pseudotype, MuSV(FLV), took place after two passages.

Purified preparations of Mycoplasmatales virus-laidlawii 3 were negatively stained and studied by electron microscopy. They were seen to consist of uniform sized particles having a polyhedral head, 57 nm by 61 nm, and a short tail, 25 nm long, joined to the head at one vertex by a collar. The particles were shown to have buoyant densities of 1.477 g/ml in CsCl, 1.32 g/ml and 1.26 g/ml in potassium tartrate and a sedimentation coefficient, s 20, w, of 290 ± 13. They are composed of 35.2% double-stranded DNA and five structural polypeptides with approximate mol. wt. of 172000, 81000, 73000, 68000 and 43000. The classification of the virus from its morphology and chemical properties is discussed.

The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have been examined by polyacrylamide gel electrophoresis. Four major polypeptides, VP1 (mol. wt. 200000), VP2 (50000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VP1a (105000) and VP1b (80500) have been reproducibly demonstrated in the virion, of which VP1, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VP1, thus identifying this glycopolypeptide as the major structural component of the projection.

Polysomes from Aedes albopictus cells were identified by their rapid labelling with radioactive amino acids and their sensitivity to EDTA, RNase and puromycin. The major ribosome component in cytoplasmic extracts had a sedimentation coefficient of approx. 95S and may be a ribosome dimer.

In Semliki Forest virus infected Aedes albopictus cells, 42S and 26S RNA were the major virus RNA species detected up to 10 h post infection. Virus RNA was detected in association with pre-labelled ribosomes and banded at a buoyant density of 1.55 g/ml. 42S, 38S, 33S and 26S virus RNA species were associated with polysomes.

Mouse and cat cells were each examined for the mode of restriction of endogenous xenotropic oncornavirus. Murine xenotropic helper virus (MuX) and its pseudo-type of Moloney murine sarcoma virus (MSV(MuX)) were grown in cat cells to high titre. MuX alone did not replicate in any mouse cell tested including normal or transformed outbred Swiss 3T3 cells or SC-1 cells, but did grow in a variety of other mammalian cells. MSV(MuX) was not able to achieve that intracellular state from which it could be rescued by mouse leukaemia virus (MuLV) in any mouse cell tested with the exception of SC-1 cells. Detection of MSV(MuX) foci with appropriate helper virus was as sensitive in SC-1 cells as in the cells of several other species. Sequential passage of MSV(MuX) virus complex in SC-1 cells resulted in a loss of infectious sarcoma and helper viruses, but transformed, MSV rescuable cells were retained.

If cat embryo cells were infected with either the feline endogenous xenotropic virus (FeX) or its MSV pseudotype (MSV(FeX)), two analogous states of restriction were observed. FeX alone did not replicate in cat cells as measured by release of progeny virus or by FeX group-specific antigen induction. Cat cells could be susceptible or insusceptible to the entry of MSV(FeX) as measured by MSV rescue with appropriate ecotropic feline leukaemia virus. The sensitivity of detection of MSV(FeX) foci in some cat cells in the presence of feline ecotropic virus was comparable to that exhibited by cells of other mammalian species. A single strain of cat cells underwent a change in its restrictive capacity for MSV(FeX) on prolonged passage. Late passage cat cells became very insusceptible to MSV(FeX) but not to other pseudotypes of MSV. Infectious FeX or its group-specific antigens were not detected in the insusceptible cells. The major glycoprotein of FeX did appear as a surface antigen of the insusceptible cells. It is apparent that two levels of cellular restriction can be distinguished in each of two mammalian cell systems by the susceptibility to penetration of MSV coated with endogenous xenotropic oncornavirus.

Sendai viruses were attached to liposomes (vesicular model membranes) at 0 to 4 °C, and were then incubated at 37 °C. Liposomes made of phosphatidylcholine, cholesterol and gangliosides enveloped the viruses at 37 °C to give a picture that resembles the ingestion step of phagocytosis. Virus particles were enveloped only by liposomes that contained gangliosides which serve as Sendai virus receptors.

In an attempt to differentiate between thymidine kinase (EC. 2.7.1.21) induced by herpesvirus hominis type 1 (TK 1) and type 2 (TK 2), the different susceptibilities to the modifying effects of some thymidine analogues proved to be useful criteria: (1) 2′-deoxythymidine-5′-triphosphate (dThd-5′-PPP) inhibits TK 2 at a concentration of 0.125 mm by 90%, whereas TK 1 is inhibited at 4.03 mm by 50%. (2) 2′-deoxythymidine-5′-monophosphate (dThd-5′-P) competitively inhibits TK 2 at all concentrations tested. On the other hand, the direction of its effect on TK 1 is concentration dependent: at 500 µm it stimulates and at 8 mm inhibits TK 1 activity. During enzyme kinetic studies, TK 1 displays substrate inhibition which is reversed by dThd-5′-P. This result explains the stimulating effect of dThd-5′-P at 500 µm. This phenomenon suggests the existence on the enzyme molecule of a second binding site for dThd which mediates substrate inhibition and which can be occupied also by dThd-5′-P. After polyacrylamide gel electrophoresis of TK 1, the stimulation by dThd-5′-P disappears, suggesting the separation of the second binding site from the catalytic centre.

A procedure was developed to isolate tomato mesophyll protoplasts in amounts sufficient for studying virus infection and multiplication. The infection of protoplasts with a tomato strain of tobacco mosaic virus occurred most efficiently when the inoculum contained 1 µg/ml virus, 1 µg/ml poly-l-ornithine and 0.01 m-phosphate buffer, pH 6.7. The efficiency of infection was influenced by the concentration of ions as well as by the pH of inoculation buffer. The virus infected protoplasts of plants homozygous for the gene Tm-2a, which is responsible for TMV-resistance of intact leaves, and multiplied as rapidly in them as in protoplasts from the susceptible parent line. In TMV-inoculated leaf discs prepared from the resistant plants, however, the growth of virus was very limited when compared with the large yield of virus obtained from leaf discs of the susceptible plants. The possible nature of the resistance is discussed in the light of these findings.

Protoplasts isolated from Nicotiana tabacum (cv. Xanthi) leaves were infected with cucumber green mottle mosaic virus (CGMMV). Progeny CGMMV particles were first detected in the protoplasts at 24 h after inoculation. They were in small crystalline arrays confined in the ground cytoplasm. The earliest detectable cytological modification in the protoplasts was the presence of peripheral vesicles in the mitochondria at 24 h after inoculation. No other effects on the cytoplasm or organelles were noted.

The multiplication of a temperature-sensitive (ts) mutant of cowpea chlorotic mottle virus (CCMV) in tobacco protoplasts has been examined. The results were similar to those obtained with intact cowpea plants. No intact virus was produced above 33.5 °C, and no coat protein, either soluble or insoluble was detected in the absence of intact virus. Ribonucleic acid (RNA) of the ts mutant, however, was synthesized at 35 °C. Virus particles made at 25 °C were degraded in vivo when the infected protoplasts were subsequently cultured at 35 °C. RNA-3 of ts CCMV was apparently encapsidated much more often than RNA-3 of wild-type (wt) CCMV in doubly infected protoplasts cultured at either 25 or 35 °C.

Eighteen new ts mutants of poliovirus have been isolated after a variety of mutagenic treatments, and their loci identified in relation to the previous genetic map. The map was only extended by 25%, and the physiological characters of the new isolates corresponded in all aspects tested with those of the previous isolates. Apparently single mutants at the extreme left of the map were defective in synthesis of both double- and single-stranded RNA, functions that do not co-vary in other mutants. Two procedures respectively predicted to induce mutations preferentially in the 5′ and 3′ regions of the genome gave isolates which all indicated that the structural protein region was nearest the 5′ end. The loci for resistance to dextran sulphate and to ethyl-2-methylthio-4-methyl-5-pyrimidine carboxylate both lie in the structural protein region.

Protein obtained by degradation of particles of the ‘Alliaria’ strain of TuMV (AlTuMV) was heterogeneous. Polyacrylamide gel electrophoresis experiments showed that this heterogeneity is greatly increased when purified virus suspensions are stored for 60 h in 0.02 m-borate buffer, pH 7.5, at 4 °C instead of being immediately degraded with 1% sodium dodecyl sulphate (SDS). When freshly purified virus particles were degraded, the protein preparations contained three components, I, II and III (estimated mol. wt. 38000, 30000 and 27500, respectively) whose relative amounts differed between samples of virus. It is suggested that components II and III, which both react against AlTuMV antiserum, are produced by degradation of component I. Electrophoresis in the presence of urea at different gel concentrations indicated that components I, II and III observed in the polyacrylamide-SDS system differ both in mol. wt. and in charge density.

Rous-associated virus-61 was used as a model for studying the labelling specificity of the chloramine-T iodination procedure when applied to intact enveloped viruses. The specificity of labelling depended markedly on the concentration of iodide in the reaction mixture. At low concentrations of iodide (below 0.5 µm) only the surface proteins and lipid of intact virions were iodinated; there was no detectable labelling of internal proteins. At 10 µm-iodide, however, both internal and external proteins were iodinated; moreover there was a marked change in the reactivity of the surface proteins. It appears that the lipid envelope provides an effective barrier to the iodinating complex generated at low, but not at high, concentrations of iodide. These and other observations suggest that the chloramine-T procedure has a previously unrecognized potential for specifically labelling the surface proteins of lipid-enveloped structures.

Pike fry rhabdovirus (PFR) RNA has been characterized as a single-stranded non-segmented molecule of about 4 × 106 daltons. In sucrose gradients in 0.1 m-NaCl it has a sedimentation coefficient of 40 to 45 S, somewhat lower than that of Semliki Forest virus RNA. The sedimentation velocity of the RNA is strongly influenced by the salt concentration and divalent cations. It shows a buoyant density of 1.65 g/ml in caesium sulphate. The base composition of the RNA is 21.6% G, 25.1% A, 22.4% C and 30.9% U. It is thus comparable to the RNAs of other rhabdoviruses.

An RNA-synthesizing complex was found in the nucleoplasm of fowl plague virus-infected chicken fibroblast and Ehrlich tumour cells. The complex sedimented at 120 S and banded in caesium chloride at 1.39 to 1.41 g/ml. It contained an influenza nucleocapsid protein as a major protein constituent. The complex functioned late in infection, and RNA synthesis in it was resistant to actinomycin D, the properties expected of influenza virus replicative complex.