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J. gen. Virol. (1977) , 36, ~ 541-545 54~ a i d Printed in Great Britain The Circular, Segmented Nucleocaspid of an Arenavirus-Tacaribe Virus (Accepted 12 M a y 1977) SUMMARY The nucleocapsid structures of Tacaribe virus, a member of the ,4renaviridae, were purified from detergent-treated virus particles by equilibrium density gradient centrifugation. Negative-contrast electron microscopy indicated that they were coiled, circular filaments. They had a mean diam. of 5 to IO nm and two predominant length classes of 640 nm and 13oo nm were found. All viruses currently classified as members of the Arenaviridae have been shown to be morphologically identical (Rowe et al. I97O; Pfau et aL 1974; Murphy, 1975). They are spherical or pleomorphic, with an average diam. of about 12o nm. However, individual particles may vary from 50 nm to over 300 nm in diam. The virus particles have an envelope which is derived from the host cell plasma membrane and from which club-like projections radiate (Murphy & Whitfield, 1975). Thin-sectioned preparations of arenavirus-infected cells also show that these viruses contain varying numbers of electron-dense granules (now known to be ribosomes) within an unstructured interior (Murphy, I975; Rawls & Buchmeier, 1975). Neither thin-sectioning nor negative contrast electron microscopic studies have contributed to the further characterization of the internal component(s) of arenaviruses. The nucleic acid extracted from purified arenavirus preparations was single-stranded RNA and consisted of several species (Rawls & Buchmeier, 1975). For Pichinde, lymphocytic choriomeningitis (LCM) and Junin viruses, the RNA species were found to have sedimentation coefficients of 31 to 33S, 28S, 22 to 25S, I8S and 4 to 6S (Carter, Biswal & Rawls, 1973; Pedersen, 1973; Anon et aL I976). The 28S, I8S and 4 to 6S RNA species were determined to be of host ribosomal origin by base composition, methylation ratio and inhibitor studies (Rawls & Buchmeier, I975). It is currently surmised, therefore, that only the 31 to 33S and 22 to 25S RNA species are virus-specific. Recently, however, another smaller RNA species, I5S, was found in Pichinde virus (Farber & Rawls, 1975). In this paper we present data which demonstrate that the internal nucleocapsid structure of Tacaribe (TAC) virus, an arenavirus, is composed of coiled filaments which are circular. Tacaribe virus strain TVRL I 1573 (Downs et aL 1963) was plaque purified three times in continuous African green monkey kidney (VERO) cell culture monolayers which contained less than seven plaques per culture. Roller bottle cultures (490 cm2) of confluent VERO or baby hamster kidney (BHK-2I) cell monolayers, grown at 36 °C, were infected at an input multiplicity ofo.oI to o.I p.f.u./cell. Radiolabelled virus was grown at 32. 5 °C in a modified Eagle's minimum essential medium which was added 4 h after infection and contained onetenth the standard amino acids concentration and was supplemented with 3H-uridine (5 #Ci/ml) and a uniform 14C-amino acids mixture (0-2/zCi/ml). After 48 h growth, when the infectivity titre was lO7 to lO8 p.f.u./ml., the infectious cell culture fluids were clarified by centrifugation at IOOOOg for 30 min and the virus was concentrated by NaCl-polyethylene glycol precipitation (Obijeski et al. I976a). Virus particles were purified by sequential Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 18:54:54 Short communications ~1~ 542 (a) 20 1.40 16 1'30 .~ 12 1-20 ~ 1'10 x 4- -r i - ."7 /D. (b) .~ "~ "~ o 20 9~ 16 12 ! Il 4 c ~ . . . t . ' 4 8 ~ - O - O - 12 -UI-E~ ~ 16 . TOP Fraction number Fig. 1. CsC1 equilibrium density gradient centrifugation of untreated or Nonidet-P4o (NP-4o) treated TAC virus. Purified TAC virus, differentially labelled with laC-amino acids ([] - - - []) and aH-uridine ( O - - O ) , was incubated (b) with or (a) without NP-4o (2 ~ , v/v, 15 min, 4 °C). Each mixture (o'5 ml) was loaded on to separate pre-formed linear CsC1 gradients ( p = I.I to I'5 g/ml) in TSE buffer (o'o5 M-tris-HC1, pH 7"8, o'15 M-NaC1, 0"o03 M-EDTA), and the gradients were centrifuged at 45000 rev/min for 24 h at 6 °C in a Beckman SW 5o'1 rotor. After centrifugation, fractions were collected from the bottom of the tube and were weighed to determine their density ( • _ _ A ) . The distribution of radioactivity of o.I ml samples of each fraction was monitored by liquid scintillation spectrometry. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 18:54:54 Short communications 543 Fig. 2. (a) Survey electron micrograph of TAC nucleocapsid structures from virus treated with NP-4o and purified by CsCl equilibrium density gradient centrifugation. Numerous circular coiled strands were evident. At least two size classes of circles were present. (b) Higher magnifications of the two predominant size classes o f T A C nucleocapsid from which length measurements were made. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 18:54:54 544 Short communications equilibrium centrifugation in combination gradients of potassium tartrate and glycerol (KT: GLY) followed by velocity sedimentation in sucrose density gradients as described previously (Obijeski et al. I976a). Virus material was prepared for electron microscopy by a pseudoreplica technique and was stained with 0"5 ~ aqueous uranyl acetate (Palmer, Martin & Gary, I975). Preliminary negative-contrast electron microscopic studies of TAC virus purified by KT:GLY gradient centrifugation indicated that the virus particles had an electron-dense centre surrounded by an electron-lucent envelope which had club-like projections. In many of our preparations, when the negative-contrast medium penetrated the virus particle, we observed electron-dense material inside the particles that appeared to be supercoiled strands of indeterminate length (data not shown). These initial studies immediately suggested to us that TAC virus might contain a helical nucleocapsid structure. Therefore, experiments were designed to assess these observations more fully. When purified preparations of TAC virus differentially labelled with 14C-amino acids and 3H-uridine were centrifuged to equilibrium in caesium chloride, the virus particles banded at a density of 1.2o g/ml (Fig. I a). However, when a sample of the same TAC preparation was treated with Nonidet-P4o (NP-4o), virus material containing both isotopes was recovered at a density of I'3r g/ml (Fig. I b). Electron microscopy of this material (Fig. 2a) revealed well-dispersed filaments that appeared supercoiled and twisted and were 5 to ro nm in diameter. Most of the filaments could be followed over their entire length and were closed circles. Some were oriented so that length measurements could be calculated. Length distribution analyses from fifty measurements indicated two predominant size modes, at 640 nm and I3oo nm. A higher magnification of these two size classes ofTAC nucleocapsid is shown in Fig. 2 (b) where the coiled filaments appeared as' strings of beads'. The length distributions of TAC nucleocapsids did not exhibit a simple relationship to the sizes of virus-specific RNAs reported for other arenaviruses (Rawls & Buchmeier, I975). This disparity might be due to a supercoiled arrangement of the nucleocapsid which would make length measurements difficult. The circular nucleocapsid structures of TAC virus were not typical helices as reported for the nucleocapsids of other RNA-containing viruses such as the rhabdoviruses (Wagner, I975) and the paramyxoviruses (Choppin & Compans, I975). In all our TAC nucleocapsid preparations we observed some large aggregates of coiled filaments with free ends. However, it was impossible to determine whether these were unique nucleocapsid species or whether they were simple aggregates of disrupted circular species. Host cell ribosomes associated with the internal component(s) of other arenaviruses (Rawls & Buchmeier, I975) were never observed in any of our TAC nucleocapsid preparations. These findings suggest that the internal structure of TAC virus is composed of endless coiled strands which are arranged to give a circular structure. This observation does not stand alone. Recently, several members of the Bunyaviridae (Porterfield et al. I975/76)that also contain a segmented single-stranded RNA genome were reported to possess circular nucleocapsids (Pettersson &von Bonsdorff, I975; Samso, Bouloy & Hannoun, I975; Obijeski et al. I976 b). However, with La Crosse virus, detailed chemical, enzymic and electron microscopic evidence (Dahlberg, Obijeski & Korb, I977; Obijeski et al. I976b) revealed that although the nucleocapsids of this virus could be recovered as circular moieties, the RNA genome of the virus did not exist as covalently closed circles. It will be equally important to determine if the RNA molecules in TAC nucleocapsids are circular. The significance of circular, segmented nucleocapsids found in RNA viruses is unknown. Perhaps the circularity of the nucleocapsids is an important mechanism whereby the long RNA genome of the virus is accommodated in the nascent virus particle. Although not Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 18:54:54 Short communications 545 shown in this report, we have also isolated circular nucleocapsid structures from Tamiami virus, another member of the Arenaviridae. E. L. Virology Division Bureau of Laboratories Center for Disease Control Public Health Service U.S. Department of Health, Education and Welfare Atlanta, Georgia 3o333, U.S.A. PALMER J. F. OBIJESKI PATRICIA A . WEBB K. M. JOHNSON REFERENCES ANON, M. C., GRAtS, O., MAXTINEZ SEGOVlA, Z. M. & fERNANDEZ, M. T. r. (1976). R N A composition of Junin virus. Journal of Virology x8, 833-838. CARTER, M. r., BISW~L, N. & RAWLS, W. r. (1973). Characterization of nucleic acid of Pichinde virus. Journal of Virology Ix, 61-68. CHOPPIN, P.W. & COMPANS, R.W. (1975). Reproduction of paramyxoviruses. Comprehensive Virology 4, 95-178. DAHLBERG, J. E., OBIJESKI, J. F. & KORB, J. (1977). Electron microscopy of the segmented R N A genome of La Crosse virus: absence of circular molecules. Journal of Virology 22, 203-209. DOWNS, W. G., ANDERSON, C. R., SPENCE, L., A1TKEN, T. H. G. & GREENHALL, A. H. (1963). Tacaribe virus, a new agent isolated f r o m Artibeus bats and mosquitoes in Trinidad, West Indies. American Journal of Tropical Medicine and Hygiene i2, 640-646. FARBER, F. E. & RAWLS, W. E. (1975). Isolation of ribosome-like structures f r o m Pichinde virus. Journal of General Virology 26, 21-3I. MtmPHy, r. A. (1975). Arenavirus taxonomy: a review. Bulletin of the World Health Organization 52, 389-391. MURPHY, r. A. & WHITI~IELD, S. G. (1975). Morphology and morphogenesis of arenaviruses. Bulletin of the World Health Organization 52, 4o9-419. OBIJESKI, J. F., BISHOP, D. H. L., MURPHY, F. A. & PALMER, E. L. (1976a). Structural proteins of La Crosse virus. Journal of Virology x9, 985-997. OBIJESKI, J. F., BISHOP, D. H. L., PALMER, E. L. & MURPHY, F. A. (I976b). Segmented genome and nucleocapsid o f La Crosse virus. Journal of Virology 2o, 664-675. PALMER, E. L., MARTIN, M. L. & GARY, G. V¢., JUN. (1975). The ultrastructure of disrupted herpesvirus nucleocapsids. Virology 65, 26o-265. PEDERSEN, I. R. (1973). Different classes of ribonucleic acid isolated f r o m lymphocytic choriomeningitis virus. Journal of Virology IX, 416--423 . PETTERSSON, R.F. & VON BONSDORFF, C.H. (1975). Ribonucleoproteins of Uukuniemi virus are circular. Journal of Virology 15, 386-392. PFAL1, C. J., BERGOLD, G. H.~ CASALS, J., JOHNSON, K. M., MURPHY, F. A., PEDERSEN, I. R., RAWLS, W. E., ROWEr W. P., WEBB, P. A. & WEISSENBACKER,M. C. (1974). Arenaviruses. Intervirology 4, 2o7-213. PORTERFIELD, J. S., CASALS,J., CHU'MAKOV~M. P., GAIDAMOVICH,S. Y., HANNOUN,C., HOLMES, I. H., HORZINEK~ M. C., MLrSSGAY, M., OKERBLOM, N. & RUSSELL, P. K. (1975/76). Bunyaviruses and Bunyaviridae. Intervirology 6, 13-24. RAVELS, W. E. & BOCHMEIER, i . (1975). Arenaviruses: purification and physiochemical nature. Bulletin of the Worm Health Organization 52, 393-4Ol. ROWE, W. P., MURPHY, F. A., BERGOLD, G. FI., CASALS, J., HOTCHIN, J., JOHNSON, K. M., LEHMANN-GRUBE,E.~ MIMS, C. A., TRAUB, E. & WEBB, P. A. (I970). Arenoviruses: proposed name for a newly defined virus group. Journal of Virology 5, 651-652. SAMSO, A., BOULOY,M. & HANNOUN,C. (1975). Pr6sence de ribonucleoprot6ines circulaires dans le virus L u m b o (Bunyavirus). Comptes rendus hebdomadaire des Sdances de t'Acaddmie des Sciences 28o, 213-215WAGNER, R. R. (1975). Reproduction of rhabdoviruses. Comprehensive Virology 4, 1-93. (Received 2I March t977) 35 Vlrt 36 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 18:54:54