Download The Circular, Segmented Nucleocaspid of an Arenavirus

J. gen. Virol. (1977) , 36, ~
541-545
54~ a i d
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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
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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.
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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.
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544
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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
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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
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