VIROLOGY, Mar. in U.S.A. Replication of Simian Foamy in ......
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JOURNAL OF VIROLOGY, Mar. 1969, p. 358-362 Vol. 3, No. 3 Copyright © 1969 American Society for Microbiology Printed in U.S.A. Replication of Simian Foamy Virus in Monkey Kidney Cells J. K. CLARKE, F. W. GAY, AND J. J. ATTRIDGE Departmeiit of Microbiology, The Queeni's University of Belfast, Belfast BT12 6BN, Nortilern Irelanid Received for publication 29 November 1968 The structure of foamy virus, its mode of maturation, and the origin of vacuoles in monkey kidney cells are described. The morphology of a virus (strain MK5) ob- tained from rhesus monkey kidney cultures and propagated in HEp-2 cells has been described by use of ultrathin sections (2) and negative staining techniques (1). During the latter investigation, the agent was shown to be a type 1 foamy virus; since this virus is a frequent contaminant of rhesus monkey kidney cultures (4), it seemed worthwhile to examine its mode of replication in its natural host tissue in order to compare these results with those previously obtained in HEp-2 cells (2). This examination revealed host-depend- ent variations in the morphogenesis of the virus which are described in this communication. Cultures of primary rhesus monkey kidney cells (assumed to be free from extraneous viruses since they showed no spontaneous cytopathic effect, failed to haemadsorb chick or guinea pig erythro- cytes, and contained no agent which could be de- tected by ultrathin section) were infected with strain MK5 or a foamy virus freshly isolated from rhesus monkey kidney cultures. After 4 to 6 days, when about 50% of the monolayers showed "foamy" degeneration, the cells were scraped from the glass, fixed with 1 %, osmium tetroxide for 2 hr at 4 C, and embedded in Maraglas; the sections were then stained with uranyl acetate and lead citrate. Cells were not synchronously infected so that various stages of virus formation were found in a single preparation. Groups of the internal com- ponents of the virus were commonly seen in the cytoplasm. The internal components were 50 to 60 nm in diameter, surrounded by an ill-defined halo, and associated with a matrix which stained slightly more densely than the surrounding cyto- plasm (Fig. 1). At a presumably later stage in the process of virus maturation, the internal com- ponents were associated with extensive areas of the endoplasmic reticulum (Fig. 2), but a few were found at the plasma membrane. They matured by budding (Fig. 2, arrow) and thus acquired an envelope derived from a cell mem- brane. Although the budding process could be initiated in association with morphologically normal endoplasmic reticulum, the endoplasmic reticulum tended to lose ribosomes and become dilated to form vacuoles (Fig. 3). In this figure, a few remaining ribosomes are indicated (upper arrows); the membrane is associated with budding virus (lower arrow), whereas the vacuoles con- tain mature particles. Maturation also occurred at the plasma membrane and at the outer nuclear membrane, thus producing complete virus out- side of the cells (Fig. 8) and between the two nuclear membranes (Fig. 4). In severely damaged cells, the cytoplasm con- tained little except large vacuoles or clear spaces traversed by membranes. These membranes were still associated with budding or mature particles (Fig. 5). A few vacuoles contained virus with damaged or incomplete envelopes (Fig. 6) which may represent lysosomal digestion. Mature particles (Fig. 7, 8) had a diameter of 90 nm and consisted of internal components enveloped by a membrane bearing projections. The triple-layered structure of the membrane was resolved in many particles, especially in those which were extracellular, even after incorporation into the virus envelope. No significant change was seen to occur in the complete particles after their release, although in a few particles the nucleoid was slightly eccentric (Fig. 7). Monkey kidney cells inoculated with foamy virus and examined by light microscopy are char- acterized by the formation of large vacuoles (3). These vacuoles are seldom seen in infected HEp-2 cells (Clarke, unpublished data) and this observa- tion is given significance by the present findings. These findings show that in MK cells virus ma- tures mainly at the endoplasmic reticulum which dilates in response to the infection thus forming vacuoles, whereas in HEp-2 cells maturation oc- curs mainly at the plasma membrane (2). The two types of infected cells differed in that the cytoplasm of MK cells usually contained 358 on July 7, 2018 by guest http://jvi.asm.org/ Downloaded from
Transcript of VIROLOGY, Mar. in U.S.A. Replication of Simian Foamy in ......
JOURNAL OF VIROLOGY, Mar. 1969, p. 358-362 Vol. 3, No. 3Copyright © 1969 American Society for Microbiology Printed in U.S.A.
Replication of Simian Foamy Virus inMonkey Kidney Cells
J. K. CLARKE, F. W. GAY, AND J. J. ATTRIDGEDepartmeiit of Microbiology, The Queeni's University of Belfast, Belfast BT12 6BN, Nortilern Irelanid
Received for publication 29 November 1968
The structure of foamy virus, its mode of maturation, and the origin of vacuolesin monkey kidney cells are described.
The morphology of a virus (strain MK5) ob-tained from rhesus monkey kidney cultures andpropagated in HEp-2 cells has been described byuse of ultrathin sections (2) and negative stainingtechniques (1). During the latter investigation, theagent was shown to be a type 1 foamy virus;since this virus is a frequent contaminant ofrhesus monkey kidney cultures (4), it seemedworthwhile to examine its mode of replication inits natural host tissue in order to compare theseresults with those previously obtained in HEp-2cells (2). This examination revealed host-depend-ent variations in the morphogenesis of the viruswhich are described in this communication.
Cultures of primary rhesus monkey kidney cells(assumed to be free from extraneous viruses sincethey showed no spontaneous cytopathic effect,failed to haemadsorb chick or guinea pig erythro-cytes, and contained no agent which could be de-tected by ultrathin section) were infected withstrain MK5 or a foamy virus freshly isolated fromrhesus monkey kidney cultures. After 4 to 6 days,when about 50% of the monolayers showed"foamy" degeneration, the cells were scrapedfrom the glass, fixed with 1 %, osmium tetroxidefor 2 hr at 4 C, and embedded in Maraglas; thesections were then stained with uranyl acetate andlead citrate.
Cells were not synchronously infected so thatvarious stages of virus formation were found in asingle preparation. Groups of the internal com-ponents of the virus were commonly seen in thecytoplasm. The internal components were 50 to60 nm in diameter, surrounded by an ill-definedhalo, and associated with a matrix which stainedslightly more densely than the surrounding cyto-plasm (Fig. 1). At a presumably later stage in theprocess of virus maturation, the internal com-ponents were associated with extensive areas ofthe endoplasmic reticulum (Fig. 2), but a fewwere found at the plasma membrane. Theymatured by budding (Fig. 2, arrow) and thusacquired an envelope derived from a cell mem-
brane. Although the budding process could beinitiated in association with morphologicallynormal endoplasmic reticulum, the endoplasmicreticulum tended to lose ribosomes and becomedilated to form vacuoles (Fig. 3). In this figure,a few remaining ribosomes are indicated (upperarrows); the membrane is associated with buddingvirus (lower arrow), whereas the vacuoles con-tain mature particles. Maturation also occurredat the plasma membrane and at the outer nuclearmembrane, thus producing complete virus out-side of the cells (Fig. 8) and between the twonuclear membranes (Fig. 4).
In severely damaged cells, the cytoplasm con-tained little except large vacuoles or clear spacestraversed by membranes. These membranes werestill associated with budding or mature particles(Fig. 5). A few vacuoles contained virus withdamaged or incomplete envelopes (Fig. 6) whichmay represent lysosomal digestion.Mature particles (Fig. 7, 8) had a diameter of
90 nm and consisted of internal componentsenveloped by a membrane bearing projections.The triple-layered structure of the membrane wasresolved in many particles, especially in thosewhich were extracellular, even after incorporationinto the virus envelope. No significant changewas seen to occur in the complete particles aftertheir release, although in a few particles thenucleoid was slightly eccentric (Fig. 7).Monkey kidney cells inoculated with foamy
virus and examined by light microscopy are char-acterized by the formation of large vacuoles (3).These vacuoles are seldom seen in infected HEp-2cells (Clarke, unpublished data) and this observa-tion is given significance by the present findings.These findings show that in MK cells virus ma-tures mainly at the endoplasmic reticulum whichdilates in response to the infection thus formingvacuoles, whereas in HEp-2 cells maturation oc-curs mainly at the plasma membrane (2).The two types of infected cells differed in that
the cytoplasm of MK cells usually contained358
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VOL. 3,1969NOE39
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FIG. 1. Group of cytoplasmic intternial componzents is seeni in a relatively denisely stainied matrix. Bar in the
figure representts ttm. >X 30,000.
FIG. 2 Iinternial compontents are seen hin the cytoplasm associated mabdiy with the entdoplasmic reticulum bitt
also with the plasma membranie. Ani occasio,ial particle is buiddinig throuigh thte enidoplasmic reticulum (arrow).Bar hin the figuire represenits pim. X 28,000.
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FIG. 3. Mature particles (i.e., enveloped internial componenzts) are seen in "vacuoles." Tlhe outer surfaces ofthese vacuoles still retain? a few ribosomes (top arrows). A budding particle is also shown (lower arrow). Bar inthe figure represents 500 nm. X 36,000.
FIG. 4. Two nuclear membranes are seeii at the left, where the internal componenit is budding from thte cyto-plasm into a space between tltem to form a mature particle. Free internal components are seen in the cytoplasm,and a group qf mature particles is visible (center) between the nuclear membrantes. Bar in the figure represents1 um. X 72,000.
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7 -,8-FIG. 5. Cytoplasm of a degenerated cell is shtown; thle membranes are associated with buddinig and mature
particles. Bar in thefigure represents 500 rnm. X 40,000.FIG. 6. Vacuole which is filled with mature particles is sh1own. Th7e envelope of mali)' of these particles is dam-
aged or incomplete. Bar in the figure represents 500 trm. X 50,000.FIG. 7. Extracellular mature particles are shown. Thle two particles on the left lack projections, and the ullit
membrante structure of the entvelope is seeni. Thlis is visible, though less distinict, in the particles which possess pro-jections. Bar in thle figure represenits 100 mzm. X 100,000.
FIG. 8. Mature extracellular particles are seemi. Thle unlit membranie structlure of thte emuvelope is visible evenin the presemice of projectiomus. Bar in thte figure represenits 100 mnm. X 100,000.
361
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362 NOTES
many internal components but no inclusions,whereas in HEp-2 cells relatively few internalcomponents were seen and these tended to beassociated with large accumulations of granularmaterial (2). The structure of the membrane of thevirus envelope also appeared to depend on thehost tissue, thus, in contrast to the present find-ings it was seen as a single line in HEp-2-grownvirus (2). It is possible that the triple-layeredstage may represent only an intermediate stage ofthe formation of the envelope, but since theunit membrane was best resolved in extracellularparticles, i.e., in what is presumably relatively"old" virus, this is unlikely.
It was suggested that strain MK5 resembledBittner virus in some respects (2), but the per-sistent failure to demonstrate a double-shell
J. VIROL.
structure in the internal component of MK5makes it likely that the two agents belong todistinct virus groups.
We thank K. B. Fraser for advice and P. Clyde for technicalassistance.
This investigation was supported by the National Fund forResearch into Crippling Diseases.
LITERATURE CITED
1. Clarke, J. K., and J. T. Attridge. 1968. The morphology ofsimian foamy agents. J. Gen. Virol. 3:185-190.
2. Clarke, J. K., J. T. Attridge, and F. W. Gay. 1969. The morpho-genesis of simian foamy agents. J. Gen. Virol. 4:183-188.
3. Plummer, G. 1962. Foamy virus of monkeys. J. Gen. Micro-biol. 29:703-709.
4. Stiles, G. E., J. L. Bittle, and V. J. Cabasso. 1964. Compari-son of simian foamy virus strains including a new serologicaltype. Nature 201:1350-1351.
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