Arenaviruses are enveloped.
Cryo-EM revealed that Pic, Tac, and LCM are roughly spherical virions in which the lipid bilayer is studded with the viral glycoprotein spikes (Fig. ). The center-to-center distance between the maximum phospholipid headgroup density of the inner and outer bilayer leaflets of virions and copurified smooth-walled vesicles was about 35 Å. This value was reasonable given that the maximal densities in most common cellular lipid bilayers are separated by ~36 to 40 Å (35
). The bilayer density was used as a fiducial mark throughout these studies. The closeness of observed and expected sizes was taken as an indication that densities visible in these images could be measured to within a few angstroms.
Electron cryomicroscopy of arenaviruses. Images of native frozen-hydrated Pic (A), Tac (B), and LCM (C) virions are shown. Bar, 500 Å.
Arenaviruses have a large variation in diameter.
The diameters of all Pic, Tac, or LCM virions ranged from 400 to 2,000 Å (Fig. ). The mean virion diameters (± SD) were 840 ± 200 Å for Pic (n
= 407), 920 ± 200 Å for Tac (n
= 308), and 860 ± 210 Å for LCM (n
= 337). Interestingly, the diameter histograms displayed at least four peaks for LCM separated by 130 ± 20 Å (n
= 18), with diameters measuring 520 ± 50, 650 ± 20, 780 ± 10, and 900 ± 30 Å (n
= 3 each). This pattern was less pronounced for the Pic and Tac particles. The appearance and spacing of the peaks were not affected when the LCM sample size was increased to ~750 virions. Most virions clustered around the 780- and 900-Å diameters. The mean virion diameters were slightly smaller than those reported for arenaviruses grown in cell culture and in infected tissue, as observed by thin-section electron microscopy, negative-stain transmission electron microscopy, and scanning electron microscopy (reviewed in reference 26
). Arenaviruses do not display the prominent seams observed in retroviral capsids (18
FIG. 2. Comparison of arenavirus morphology and size distribution. Electron micrographs of frozen-hydrated virions of Pic (A and F), Tac (B), LCM (C), 1 M LiCl-treated LCM (D), and 1 M NaCl- and pH 5.0-treated LCM (E) are shown. The images in panels A to E were (more ...) GP-1 and GP-2 associate by electrostatic interactions.
The glycoprotein spike (GP-C) consists of a peripheral domain (GP-1) and an integral membrane protein domain (GP-2). To examine the role of electrostatic interactions in the association of GP-1 and GP-2, purified, radiolabeled virions were incubated in high-ionic-strength buffers (9
). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose gradient fractions showed that GP-1 was released from Pic, Tac, and LCM virions following incubation in 1 M LiCl at neutral pH or in 1 M NaCl at pH 5.0 for 1 h at 37°C (data not shown). Although Tac GP-1 and GP-2 displayed the same electrophoretic mobility, they could be distinguished by Western immunoblot analysis (25
). Consistent with these results, high-salt- and low-pH-treated LCM virions lacked visible surface projections, confirming that GP-1 accounts for the ectodomain of GP-C (Fig. ).
Layers of density are closely apposed to the inner bilayer leaflet.
Rotationally averaged radial density profiles of the most circular virions displayed a thin, concentric layer of density (designated inner track 1 [IT-1]) that was closely adherent to the inner bilayer leaflet (Fig. ). The maximum density of IT-1 was located 46 ± 4 Å (Pic), 39 ± 4 Å (Tac), or 43 ± 5 Å (LCM) (n = 12 for each) radially inward from the maximum phospholipid headgroup density of the inner bilayer leaflet. Virions also displayed a second concentric inner track (IT-2) with a maximum density located 77 ± 6 Å (Pic), 105 ± 8 Å (Tac), or 78 ± 3 Å (LCM) (n = 12 for each) radially inward from the maximum headgroup density of the inner leaflet. LCM and Pic virions were indistinguishable, while Tacaribe virions were recognizable by the increased distance separating IT-1 from IT-2. The IT-2 layer was formed by ~50-Å globular densities. Occasional threads of density appeared to connect the IT-2 density and GP domains (Fig. ), but these features were not uniformly seen. Class averages indicated that no features were reproducibly located interior to IT-2.
FIG. 3. Structural features of Pic, Tac, and LCM arenaviruses revealed by rotationally averaged radial density profiles. Twelve well-centered, circular virion projections displaying bilayer leaflets of approximately even intensity were aligned and averaged to (more ...)
FIG. 4. Surface features as well as inner densities (IT-1, IT-2) revealed by analysis of side views. Class averages of side views reveal the lipid bilayer (LB) and GP-C ectodomain as well as inner densities. A single globular density (D, bottom) or double-lobed (more ...)
Rotationally averaged profiles of spherical virions ranging in diameter from 400 to 2,000 Å showed that the locations of IT-1 and IT-2 were independent of virion diameter for Pic and LCM. Tac virions smaller than 560 Å were rare, but inner features of the smallest Tac virions resembled the tracks for larger diameter virions. Radial density profiles of empty vesicles did not display any interior densities, thus confirming that the observed features were not created by defocus artifacts of the bilayer.
The large GP-C domains of Pic, Tac, and LCM are of comparable size.
For those particles in which the GP-C spikes were visible, the centers of the GP-C head domains were spaced 100 ± 7 Å (n = 33) apart. With this spacing, virions ranging from 400 to 2,000 Å would be expected to contain ~50 to 1,400 GP-C oligomers apiece. The spikes of Pic, Tac, and LCM extended radially from the maximum phospholipid headgroup density of the outer bilayer leaflet 90 ± 7, 90 ± 8, and 91 ± 8 Å (n = 100), respectively. GP-C ectodomain sizes did not differ significantly by the two-tailed Mann-Whitney test.
Two-dimensional averages of side views revealed two types of images, one with uniform GP-C density and a second with split density (Fig. ). The split-density GP-C head and stalk regions of LCM class averages measured 78 ± 7 by 60 ± 2 and 43 ± 5 by 33 ± 2 Å (width parallel to the bilayer by height perpendicular from the bilayer), respectively. The corresponding uniform-density head and stalk regions measured 67 ± 4 by 60 ± 4 and 30 ± 6 by 34 ± 3 Å, respectively (n
= 10 each). These two views most likely represented projections of the GP-C complex rotated about an axis perpendicular to the membrane plane. The split-density pattern and corresponding difference in the observed width of GP-C suggested C2 symmetry, but a trimeric GP stoichiometry could not be ruled out. It is also notable that split- and uniform-density images were also observed for averages of side views of the ectodomain of the Escherichia coli
ATP synthase, which has pseudohexameric symmetry (6
The estimated volume of two stacked elliptic cylinders corresponding to the head and stalk of GP-C was 2.2 × 105
to 3.5 × 105
. The boundary of the ectodomain is known from sequencing of the proteolysis-resistant C-terminal fragment of GP-C (4
). Using a partial specific volume of 0.73 cm3
), the expected volume of a monomeric, nonglycosylated GP-C ectodomain is ~5.9 × 104
. The estimated volume of the GP-C ectodomain is therefore consistent with four to six nonglycosylated GP-C monomers (Table ).
Paracrystalline packing of densities in en face views.
The observed en face density should arise from the average of the near and far sides of the envelope as well as the interior density. We reasoned that if the density within the virion core was randomly distributed, then analysis of en face views could reveal information on the packing of proteins proximal to the envelope. Images of LCM, Pic, Tac, centers of empty copurified vesicles, or regions of background vitreous ice were aligned and averaged separately by using a reference-free method. The arenavirus images showed a grid-like arrangement of ~50-Å punctate densities, while no regularly occurring features were visible in vesicle and ice control class averages. Computed diffraction patterns of the arenavirus averages displayed reflections not seen in the transform of the background ice average (data not shown). This suggested that the GP-C and/or the underlying NP molecules were packed in a paracrystalline fashion. En face images of single virions (Fig. , insets) showed similar punctate densities, and computed diffraction patterns showed discrete sampling (Fig. ). To further delineate this apparent lattice, diffraction patterns were computed for groups of 3,000 to 10,000 images of Pic, Tac, LCM, and LiCl-treated LCM that had been aligned to a reference-free class average (Fig. ). Reflections that corresponded to a lattice with a = 74 ± 5 Å, b = 57 ± 4 Å, and γ = 76 ± 3° were consistently observed (Fig. ). Image analysis of LiCl-treated or acid- and NaCl-treated LCM virions yielded an identical lattice (Fig. ). Since these virions lack GP-C spikes, the lattice in these virions must arise from one of the other structural proteins, such as Z or NP. The sizes of the roughly circular densities in the averaged images ranged from 48 to 56 Å, which is comparable to the IT-2 densities in side views of Pic, Tac, and LCM (Fig. ).
FIG. 5. Paracrystalline lattice of NP revealed by analysis of en face images. Computed diffraction patterns of class averages (large images) were constructed for en face views from Pic (A and E), Tac (B and F), LCM (C and G), and LiCl-treated LCM (D and H). Images (more ...) The IT-2 density is assigned to NP.
Volume estimations for ellipsoids, using the side (4.4 × 104
to 9.2 × 104
[Fig. ]) or en face (5.0 × 104
to 9.7 × 104
[Fig. ]) dimensions of the IT-2 and en face densities, were comparable to the calculated volume of NP (7.6 × 104
), using a protein partial specific volume of 0.73 cm3
/g (Table ). The observed size and shape of the IT-2 and en face densities resembled the 40- to 50-Å-diameter Pic RNP globules spaced 60 Å apart (50
) or 30- to 40-Å beads arranged 40 to 50 Å apart observed for both Tac and Tamiami (20
) imaged by negative-stain transmission EM on disrupted virions. The maximal center-to-center separations in the en face grid (74 ± 5 Å [Fig. ]) and the mean of the largest one-third of the IT-2 center-to-center measurements (75 ± 3 Å [n
= 25 of 74 total]) were similar. The predicted volumes of one IT-2 or en face density were comparable to that calculated for one NP molecule. Consequently, we infer that each IT-2 and en face density represents one copy of NP.
The IT-1 density is assigned to protein Z.
Side views showed that the IT-1 densities were much smaller than the IT-2 densities, with an average height of 27 ± 2 Å (n
= 18), width of 43 ± 5 Å (n
= 30), and center-to-center spacing of 90 ± 7 Å (n
= 33). Split or uniform IT-1 densities were located beneath each GP-C protrusion in class averages (Fig. and data not shown). Candidates for the IT-1 density include the cytoplasmic tail of GP-2 and Z or contributions of both. Initial characterization of the topology of the hydrophobic GP-C-signal protein in the endoplasmic reticulum (13
) appears to exclude the signal protein from consideration for IT-1. The posttransmembrane carboxyl-terminal tail of GP-2 is relatively short, between 42 and 46 amino acid residues. Thus, the main contribution to the IT-1 density is likely to be the remaining high-copy structural protein, Z. The Z protein contains no canonical hydrophobic transmembrane regions but has been shown to be tightly associated with membranes (43
) and the virion core (40
) and is also myristoylated. These features support a membrane-proximal location of Z (Fig. ). The central density of IT-1 was a reasonable volume match for two copies of Z, although it was difficult to measure the dimensions of IT-1 and the poorly resolved connecting densities.