Vol. 16, No. 4

JOURNAL OF VIROLOGY, Oct. 1975, p. 1027-1038

Printed in U.S.A.

Copyright 0 1975 American Society for Microbiology

Comparative Ultrastructural Study of Four Reticuloendotheliosis Viruses CHIL-YONG KANG,* TIMOTHY C. WONG, AND KATHRYN V. HOLMES Department of Microbiology, The University of Texas Southwestern Medical School, Dallas, Texas 75235 Received for publication 16 June 1975

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are spherical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosissarcoma viruses.

The reticuloendotheliosis group of avian viruses (REV) includes four viruses: duck spleen necrosis virus (DSNV), duck infectious anemia virus (DIAV), reticuloendotheliosis virus strain T (REV-T), and chicken syncytial virus (CSV) (7, 13, 21, 23, 25). These REVs have been isolated from several different avian species in association with a variety of syndromes including spleen necrosis, anemia, visceral reticuloendotheliosis, and infiltrative lymphoid nerve lesions (H. G. Purchase and R. L. Witter, Curr. Top. Microbiol. Immunol., in press; 7, 13, 21, 23, 25). At the present time, these REVs can be distinguished only by differences in pathogenicity (Purchase and Witter, in press; 21). Infection of cultured chicken and duck embryo fibroblasts with any REV results in the death of some cells and the establishment of a persistently infected carrier culture by about day 2 after infection (24). Previous studies indicated that REV virions are type C particles (7, 21, 26) which contain a high-molecularweight, single-stranded RNA genome (10, 16) and endogenous RNA-directed DNA polymerase activity (9a, 20, 25a). The nucleotide sequences of the RNA of all four REVs are closely related to each other, but distinct from that of the avian leukosis-sarcoma virus group (ALSV) (10, 11). All four REVs contain cross-reacting antigens including DNA poly-

merase (Purchase and Witter, in press; 17, 18) and REV group-specific antigens (H. R. Bose, personal communication). The DNA polymerase of REVs is immunologically and biochemically distinct from the DNA polymerase of avian leukosis virus (ALV) (17, 18). This report describes ultrastructural studies on the morphology and development of four REVs in chicken embryo fibroblasts. All four REVs are type C particles which mature by budding into intracytoplasmic vesicles as well as from the plasma membranes of infected cells. Our studies suggest that REV maturation resembles that of mammalian leukemia-sarcoma viruses more closely than that of ALSVs. MATERIALS AND METHODS Cells and viruses. Primary chicken embryo fibroblasts were prepared by standard techniques (1, 10) from 12-day-old chicken embryos (SPAFAS, Norwich, Conn.) and were grown in Temin modified Eagle minimal essential medium (Schwarz/Mann) with 20% tryptose phosphate broth and 5% fetal calf serum. The cultured primary and secondary chicken embryo fibroblasts were shown to be free of ALSV by assaying DNA polymerase activity in the pelleted cell-free supernatants. The sources and the methods of propagating CSV, DIAV, DSNV and REV-T have been described previously (10). Virus production was monitored by measurement of sedimentable DNA polymerase activity and by detection of incorporation

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KANG, WONG, AND HOLMES

of [3H ]uridine into virions released into the media from infected cells. Rous-associated virus-61 (RAV-61) (9) from T. Hanafusa was propagated in H. M. Temin's laboratory in C/E, ALSV-negative, chick

helper factor-negative, ALSV group-specific antigennegative chicken cells (10). Culture fluids were harvested every 24 h from the virus-infected chicken cells and stored at -20 C. The virus was concentrated and purified as described previously (10). Concentration and partial purification of virus samples for electron microscope examination were done as follows. Culture fluids were harvested 48 to 72 h after virus infection and clarified by centrifugation at 1,500 rpm for 15 min in an International PR-2 centrifuge. Cell-free fluid (30 ml) was layered on 5 ml of 65% sucrose in TSE (0.1 M NaCl, 0.001 M EDTA, 0.02 M Tris-hydrochloride, pH 7.4). Virus was pelleted onto the sucrose cushion by centrifugation in a Beckman SW27 rotor for 90 min at 25,000 rpm. The virus on the interface was used for electron microscope examination. Electron microscope preparations. Negatively stained preparations of REVs were prepared by placing partially purified virus onto carbon-coated Formvar films on 400-mesh copper grids and staining for 1 min with 2% phosphotungstic acid (PTA), pH 6.8, or 2% uranyl acetate (UAc), pH 5.3. At 12, 24, 48 and 96 h after virus inoculation, cells were fixed with 2% glutaraldehyde for 4 min and then removed from the plates by scraping with a rubber policeman. The cells were pelleted by centrifugation at 1,500 rpm for 10 min at 5 C in an International PR-2 centrifuge. The cell pellets were postfixed with 1% osmium tetroxide, dehydrated in alcohols and propylene oxide, and embedded in Epon resin as previously described (6, 14). Thin sections prepared with a diamond knife on a Porter Blum MT 2 ultramicrotome were stained with uranyl acetate and lead citrate (6, 22) and examined with a Philips 301 electron microscope. In some experiments, after osmium postfixation cell pellets were stained en bloc with 0.5% uranyl acetate in 0.14 M veronal acetate buffer, pH 6.3 (6). Nucleic acid hybridization. REV and ALV viral RNAs were prepared from purified viruses harvested from chronically infected chicken cells by techniques previously described (10). The detailed techniques for nucleic acid hybridization in liquid phase are also published (10). DSNV [1H]DNA was prepared from a DNA polymerase reaction with purified DSNV RNA as template-primer and purified ALV DNA polymerase (10). The [3HJDNA represents over 80% of the 60 to 70S viral RNA as determined by [125I]RNA protection experiments (11). RAV-61 [8H DNA was prepared from the endogenous DNA-polymerase reaction of the purified virus as has been previously described (10).

RESULTS Absence of ALSV contamination in REV preparations. Due to the similar sedimentation and density profiles of ALSV and REV, it was

J. VIROL.

necessary to rule out the possibility of ALSV contamination in the REV preparations used for these studies. The technique of nucleic acid hybridization was used as a sensitive measure of possible virus contamination. DSNV [3H ]DNA was used to detect REVspecific nucleotide sequences in RNA from purified virus preparations. As can be seen in Fig. 1, more than 80% of DSNV [3H ]DNA hybridized with DSNV RNA, and approximately 70% of DSNV [3H ]DNA hybridized with RNAs from purified CSV, DIAV, and DSNV DNA

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FIG. 1. Kinetics of hybridization of DSNV [3HJDNA or RAV-61 [3HJDNA to RNAs of DSNV, REV-T CSV,1DIAV, and RAV-61. DSNV [3HJDNA (1,200 counts/min per sample) was annealed with 0.15 fig of RNAs of DSNV (0), REV-T (c), CSV (0), or DIA V (A). RA V-61 [3HJDNA (1,500 counts/min per sample) was annealed with a mixture of 0.15 Mg of DSNV RNA and 0.0015 ,gg of RA V-61 (-) or 0.15 ,g of DSNV RNA alone (0). The hybridizations were performed at 63 C in 25 ml of standard annealing buffer (10) sealed in 25-Ml' micropipettes. At different times samples were withdrawn and frozen at -60 C. At the end of the incubation, the extent of hybridization was determined by S1 nuclease digestion (10). The results are expressed as the proportion of total [3H]DNA hybridized at a given C,t value in 0.3 M salt (RNA concentration in moles/liter x time of incubation in seconds). Approximately 4% of both DSNV [3H]DNA and RA V-61 [3HJDNA was S1 nuclease resistant in controls incubated without RNA. This was subtracted from the raw data to obtain values plotted.

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EM OF RETICULOENDOTHELIOSIS VIRUSES

REV-T at Crt values of approximately 1 mol s/ liter. This indicates that the four REVs have a very high degree of nucleotide sequence homology, as has been published previously (10, 11). In contrast, when RAV-61 [3H]DNA was hybridized with DSNV RNA, no hybridization was detected at Crt values of approximately 7 mol *s/liter (Fig. 1). Similar results were obtained using RNAs from CSV, DIAV, and REV-T (data not shown). Thus, there is little or no nucleotide sequence homology between REVs and ALSVs, and there is no detectable ALSV contamination in the REV stocks. To estimate the sensitivity of nucleic acid hybridization to detect contaminating viruses, RAV-61 RNA mixed with DSNV RNA in a ratio of 1 to 99 was hybridized with RAV-61 [3H]DNA. Approximately 40% of RAV-61 [3H ]DNA was hybridized with the mixed RNAs at a Crt value of 7 mol s/liter (Fig. 1). The Crt value calculated on the basis of RAV-61 RNA alone would be about 7 x 10 2mol *s/liter where 40% of RAV-61 [3H]DNA was hybridized. This observation agrees with previous studies showing that the half Crt values of ALSV were 2 x 102 to 5 x 102 mol s/liter (10, 12). In the experiment with 1% ALSV RNA, at a Crt value of the mixed RNA of about 9 x 10- mol *s/liter, we detected approximately 15% hybridization. This indicates that the nucleic acid hybridization technique is sensitive enough to detect a contaminating virus which represents only 0.1% of a virus preparation. Ultrastructure of REV virions. The four members of REV were studied in concentrated preparations negatively stained with either PTA or UAc, and thin sections of virions from REV-infected cells were examined (Fig. 2). The virions were spheres 110 5 nm in diameter. UAc negative staining (Fig. 2a, e, i, and m) showed that the viral envelopes were covered with apparently hollow peplomers approximately 10 4 1.5 nm in diameter at the tip by 6 i 1 nm long. The peplomers appeared to be tapered so that at the viral membrane, the peplomers were 4 + 0.5 nm in diameter. The center-to-center spacing of the viral spikes was about 14 1 nm. We calculate that there would be approximately 100 of those peplomers per virion. When virions were negatively stained with PTA (Fig. 2b, f, j, and n) they were frequently penetrated by the stain, revealing a complex "ball of string" type of virus core enclosed within the viral envelope. Peplomers were also observed on viral envelopes stained with PTA (Fig. 2b). In thin sections of virions, a dense, fuzzy layer of peplomers was observed beyond the unit

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membrane of the viral envelope (Fig. 2c, d, g, h, k, 1, o, and p). The morphology of the virus core in thin sections appeared to change during virus maturation. Budding virus particles and viruses immediately after budding (Fig. 2c, g, k, and o) showed an electron-dense, ring-shaped core structure 73 4 nm in diameter with an electron-lucent center. In these particles, there was a regular space of about 8 nm between the outer edge of the core and the inner edge of the viral envelope. When the virions had been released from infected cells, the ring-shaped virus cores became condensed to a diameter of about 67 i 5 nm, and the electron-lucent centers disappeared (Fig. 2d, h, 1, and p). Viruses trapped between two cells or within intracellular vesicles frequently had condensed polygonal-shaped cores (Fig. 2p) which had a mottled appearance in thin sections. The condensation of the viral core resulted in a space of 14 nm between the viral envelope and the virus core. A model for the fine structural organization of REVs is presented in Fig. 3. A transverse section of a virion before condensation of the core is shown on the left and after condensation of the core on the right. Ultrastructural studies of REV maturation. Uninfected chicken embryo cells (Fig. 4c) were elongated fibroblastic cells with central nuclei. The cytoplasm contained large amounts of rough endoplasmic reticulum, numerous vesicles, and some multivesicular bodies. No virus-like particles of any kind were observed in these uninfected cultures. This agrees with the lack of contaminating virus in these cells as shown above by nucleic acid hybridization studies. The maturation of all four REVs in chicken embryo fibroblasts was studied. We observed no significant differences in the replication of REV-T, DSNV, CSV, and DIAV; therefore, in the following discussion we will consider the replication of all four REV viruses together. At 12 h after inoculation with REV a small number of mature virions were observed between the plasma membranes of adjacent cells. These may represent either inoculated or newly synthesized virus particles. We did not observe budding virus particles in these 12-h specimens. Newly synthesized, released virions would have been washed off the monolayers during preparation for electron microscopy unless the virions had adsorbed to the cell surface or had been trapped between two cells. By 24 h after infection, a number of mature virions were seen at the cytoplasmic membranes. These virions on the cytoplasmic membranes are probably prog-

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KANG, WONG, AND HOLMES

J. VIROL.

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0 FIG. 2. Comparative study of virions from four different REVs. (a-d) CSV; (e-h) DIAV; (i-I) REV-T; (m-p) DSNV. The virions of the four REVs were prepared by centrifugation onto a sucrose cushion. When virions were negatively stained with UAc (a, e, i, and m) numerous peplomers (arrows) were observed projecting from the viral envelope. Virions negatively stained with PTA (b, f, j, and n) showed peplomers (arrows) and often revealed a complex inner structure resembling a ball of string (i). When thin sections of virions from infected cells were stained with lead citrate and UAc (c, g, k, and o), the viral envelope with its dense layer of projecting peplomers (arrows) was clearly visible. A dense crescent-shaped core was visible within the virion. Thin sections of virions from cells that were stained en bloc with UAc (d, h, l, and p) showed a clearly defined unit membrane in the viral envelope. Many of these virions had been released from the cell, and the viral cores had condensed to form a compact arrangement of electron-dense strands. x 150,000.

eny viruses, since an increased percentage (about 35%) of cells had virions on the cell surface at this time. Most cells were producing virions by 48 h after inoculation. Budding virions were observed both at the plasma membrane and at smooth-walled, intracytoplasmic vesicles (Fig. 4, 5, and 6). Large numbers of mature virions with condensed cores were observed between adjacent cells (Fig. 7) and within large cytoplasmic vesicles. The probable sequence of events in

the budding of REV is illustrated in Fig. 6. The first stage in virus budding appeared to involve the assembly of electron-dense nucleocapsid material beneath an area of plasma membrane which had been altered by the addition of viral peplomers to the outer leaflets of the plasma membrane. Next, the virus bud elongated with the addition of dense material to the nucleocapsid core. The nucleocapsid in the budding virions always developed into a crescent-shaped dense mass with an electrorn-lucent center

VOL. 16, 1975

EM OF RETICULOENDOTHELIOSIS VIRUSES

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FIG. 3. Diagrammatic representation of an REV virion. The left portion of the diagram shows an immature REV and the right portion shows a mature REV particle after condensation of the core.

(Fig. 6c and d), resembling the type C murine leukemia viruses. The entire envelope was covered with peplomers (Fig. 6c and d). In only one instance were peplomers seen extending continuously from the viral envelope onto the surface of the adjacent plasma membrane of the cell. REV appeared to be uniformly spherical viruses. No filamentous virus forms were seen in cells infected with any of the four REV strains. As the budding virus sphere reached full size, a constriction appeared in the plasma membrane beneath the virus. No nucleocapsid material was observed within the resulting slender stalk which connected the virion to the cell. We occasionally observed surface invaginations of the plasma membrane immediately adjacent to the stem of a budding virus (Fig. 6c). These invaginations were approximately the same size as the maturing virus particle. It appears possible that they may be involved in some way in the process of virus budding. Similar invaginations have been seen near other types of viruses which bud from the plasma membrane, including pneumonia virus of mice (6). Upon release of the virions from the cell, the cores became condensed into a dense structure which appeared to be composed of electrondense strands. Similar rearrangements of viral nucleocapsid after virus release have been re-

ported with the ALSVs and murine leukemia viruses (MuLVs) (3, 4). When large virus aggregates were trapped between two adjacent cells (Fig. 7), it was possible to see viruses at several different stages of maturation simultaneously. Occasionally aberrant forms of budding virus particles were seen. Figure 8 shows two virus particles connected to the plasma membrane of a cell by a common stalk. An unusual group of eight virions which shared a common viral envelope and apparently a single stalk was observed in a thin section of a chronically infected chicken embryo cell. It would appear from this type of image that there may be sites on the cell surface which are "hot spots" for viral maturation and that sequential virus buds may develop there without completed maturation and release of the preceding virions. Virus-induced cytopathic effect. Because our experiments were performed with a low multiplicity of infection under conditions in which multiple cycles of virus maturation may be required to infect all cells, we have not yet been able to study the early events in REV infection. Cells chronically infected with REV appear normal when observed by phase contrast microscopy. By electron microscopy, the major difference between control and REV-infected cells was the presence of a number of virions in

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Comparative ultrastructural study of four reticuloendothelias viruses.

Vol. 16, No. 4 JOURNAL OF VIROLOGY, Oct. 1975, p. 1027-1038 Printed in U.S.A. Copyright 0 1975 American Society for Microbiology Comparative Ultra...
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