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Cytoplasmic Structures Associated with an Arbovirus Infection: Loci of Viral Ribonucleic Acid Synthesis PHILIP M

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Cytoplasmic Structures Associated with an Arbovirus Infection: Loci of Viral Ribonucleic Acid Synthesis PHILIP M JOURNAL OF VIROLOGY, Nov. 1968, p. 1326-1338 Vol. 2, No. 11 Copyright @ 1968 American Society for Microbiology Printed in U.S.A. Cytoplasmic Structures Associated with an Arbovirus Infection: Loci of Viral Ribonucleic Acid Synthesis PHILIP M. GRIMLEY, IRENE K. BEREZESKY, AND ROBERT M. FRIEDMAN Pathologic Anatomy Branch and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 31 July 1968 Unique cytoplasmic structures, herein designated as type I cytopathic vacuoles (CPV-I), are found in chick embryo cells early in the logarithmic phase of Semliki Forest virus replication. High resolution autoradiography demonstrated that the CPV-I are loci of 3H-uridine incorporation. This evidence correlates well with previous biochemical data and electron microscopy of the subcellular fractions active in Semliki Forest virus ribonucleic acid synthesis. Origin of the CPV-I within host cell cytoplasm is confirmed by the distribution of electron-dense tracer par- ticles and sequential ultrastructural observations. The formation of unique intracytoplasmic sodium deoxycholate (12, 14). We demonstrated vacuoles within cells infected by Semliki Forest the participation of CPV-I in viral RNA synthesis virus (SFV) and related members of the arbovirus by means of high resolution autoradiography. group has been demonstrated by electron micros- Ultrastructural studies of the infected cells at copy in both cell cultures and the cerebral tissues sequential intervals and the distribution of an of experimental animals (21, 24, 32). The cyto- electron-dense tracer have provided additional pathic vacuoles (CPV) may be subdivided into information concerning the origin and fate of two morphologically distinct groups. (i) A type the arbovirus CPV. of CPV recently recognized in chick embryo cells infected by SFV (1, 14) or Sindbis virus (un- MATERIALS AND METHODS published data). These occur relatively early in Virus and cell cultures. The Kumba strain of the infectious cycle and are, therefore, designated Semliki Forest virus was grown at 37 C in primary CPV-I. They are characterized by a regular series cultures of chick embryo cells (15). Monolayers of of membrane buds or spherules which are at- chick embryo fibroblasts were infected at virus-cell tached to the interior surface of the vacuoles. The multiplicities of 20:1 to 500:1. Virus was titrated by buds or spherules are formed of membranes with plaque assay (16). In some experiments, virus was unit structure (1), and may contain an electron- absorbed to the cells overnight at 4 C, either in dense central spot. (ii) The type of CPV that was medium containing 1 jug of actinomycin D per ml or originally described in cells infected by Western in medium without actinomycin D. In other experi- equine encephalitis virus (23). These vacuoles are ments, virus was added directly to the monolayers, with or without actinomycin D, and maintained at encircled on the cytoplasmic aspect by closely 37 C. Cultures were studied at selected intervals (0.5 spaced particles of uniform size (25 to 30 nm). to 16 hr) after the initiation of infection. In cold (4 C) They are most numerous in the late (linear) phase virus absorption, the period of infection was con- of the virus replication cycle and are designated sidered to begin from the moment of rewarming to CPV-II. CPV-II have been observed in cell 37 C (13, 15). Eight separate experiments were con- systems infected by SFV and several other group ducted to study the ultrastructural sequence of virus A arboviruses (3, 10, 23-26). development at relatively low multiplicity of infection Preliminary studies in this laboratory showed (20:1 to 40:1). In addition, two experiments employ- that CPV-I arise the early stages of SFV ing a high virus multiplicity (500:1) were conducted during to trace virus entry and to detect early evidence of infection and are present in the cytoplasmic frac- replication (up to 3 hr). The preparation of high titer tions which incorporate 3H-uridine (14). In SFV SFV pools for experiments requiring a high virus to infection, the new viral ribonucleic acid (RNA) is cell multiplicity has been reported previously (13). closely associated with cytomembranes and may Electron microscopic preparations. Cell monolayers be released from the membrane sediment by were scraped from the petri dish with a rubber police- 1326 VOL. 2. 1 968 ARBOVIRUS INFECTION 1327 man and transferred to centrifuge tubes along with the RESULTS original culture fluid. Some of the supernatant fluid was withdrawn before centrifuging at 785 X g for Virus grouwth chlaracteristics. In the chick 5 min. The resulting pellet was fixed for I to 2 hr embryo cells treated with actinomycin D and with 3'( glutaraldehyde (4) in 0.1 M phosphate buffer incubated at 37 C with 20 plaque-forming units (pH 7.3). In experiments employing Thorotrast (PFU) of SFV per cell, the titers of virus infec- (thorium dioxide) or sH-uridine, cell monolayers were tious units increased exponentially during a period fixed directly in the petri dishes before centrifugation. of I to 5 hr (Fig. 1). After 8 hr, production of Glutaraldehyde-fixed pellets were washed in 0.1 M infectious virus reached a plateau. The rising phosphate buffer containing sucrose, prior to 1 hr of titer of cell-associated virus closely paralleled the postfixation in 1'c osmium tetroxide which was also virus in the culture both buffered with 0.1 M phosphate. After rapid dehydra- titration curve of fluid, tion in graded 70 to 100', ethyl alcohol solutions, the in slope and in maximal height. pellets were embedded in Luft's Epon formula (14). Uninfected cells. Chick embryo cells in primary Sections were cut by an automatic ultratome with culture displayed a considerable pleomorphism of diamond and glass knives. They were mounted on cytological appearance, but a majority resembled uncoated copper mesh grids, stained with 10'( uranyl fibroblasts or histiocytes. Most cells contained an acetate in methanol, and examined at original magnifi- abundance of dispersed ribosomes in the cyto- cations up to 18,000 in an RCA 3F electron micro- plasmic matrix. Aggregation of ribosomes into scope operated at 50 kv. crystalloid arrays was occasionally noted within Piulse labelinig. At indicated times after virus ilnfec- the cytoplasmic matrix or adjoining the limiting tion, the culture medium was decanted, and I ml of reticulum. Eagle's medium containing 100 Ac of :H-uridine (20 membranes of endoplasmic Cyto- c/mm) was added for 3 min. The monolayers were plasmic lipid droplets, phagocytic vacuoles, and then quickly washed five times with chilled 0.85', fenestrations were common. The latter ofter NaCl (containing uridine) and fixed immediately with measured more than 2 ,m in diameter. In cells glutaraldehyde. In control tests, monolayers were treated with actinomycin D, the nucleolar fixed with Carnoy's ethyl alcohol-ether fluid, and changes were characteristic (29), and cytoplasmic acid-precipitable radioactivity was counted with a vacuoles containing autophagic whorls of mem- Packard Tri-Carb scintillation spectrometer model brane remnants frequently appeared (9). Neither 3003 (Parkard Instrument Co., Downers Grove, 111.). control Autoradiograpliy. Thick epoxy sections (0.5 to 1.0 in treated nor untreated cells, however, ,um) for light microscopy were picked up oni 'subbed' were the arbovirus CPV ever identified. slides (2) and air-dried. They were coated with Kodak AR-10 stripping film (27) or Ilford L-4 Nuclear Research Emulsion Illford. Ltd., Ilford, Essex, Re/eosed Virus England (2)]. The procedures for electron-microscopy were based on the method of Caro et al. (2). Control -J grids without sections were covered with emulsion. exposed to room light, and examined in the electron Associoted Virus microscope with or witlhout development to check grain distribution. Other control grids with unlabeled sections or without any sections were coated, stored zZ - / - for 2 to 10 weeks, and developed to determine back- ground grains. Thick (0.5 to 1 ;Am) sections were stained through the emulsion with 0.1'( Toluidine ID Blue in aqueous solution. Thin sections were ex- J - amined in the electron microscope without staining. 0 Tlhoroirat.st tracer. In four groups of experiments, a colloidal suspension of 24 to 26%'' Thorotrast was either added to cell pellets during fixation or intro- duced inito the culture medium (I to 2 cc/10 cc of medium) for 5 to 60 min precedinig fixation (no tracer added). For the tracer-fixation procedure, some cell pellets were prefixed in 3' glutaraldehyde (4) for 5 min before thie addition of 3' glutaraldehyde con- taining I to 2:10 parts ol the volume of the 24 to 26', Thorotrast. Other pellets were fixed directly in the I_l '0 2 3 4 5 6 7 8 9 10 Thorotrast-glutaraldehyde mixture. A similar volu- HOURS AFTER INFECTION WITH SFV metric proportion of the 24 to 26' Thorotrast was FIG. 1. Titers of SFV in click embrYo cells at maintained in the phosphate buffer-sucrose rinse and various times after inf'ction, wit/i a miultiplicity of 20 in the 1 (- osmium tetroxide postfixation. plaqule-forming unlits per cell. 1328 GRIMLEY, BEREZESKY, AND FRIEDMAN J. VIROL. In.fected cells. In six different series of experi- et. al. (10) occurred in the latest stages examined ments, the chick cells were examined after infec- (16 hr). tion with 20 to 50 PFU of SFV per cell. The The cycle of virus development was asyn- earliest ultrastructural changes attributable to chronous at all multiplicities of infection studied; SFV were the occurrence of surface budding virus nevertheless, certain trends in the development particles and CPV-I at 2.5 to 3 hr. By 3 to 4 hr of cytopathic vacuoles were evident (Fig. 11). after the initiation of infection, cross sections The CPV-I were most prominent during the through 10 to 20% of cells observed in random early phase of infection.
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