Cytomegalovirus primary envelopment occurs at ... - Semantic Scholar
JVI Accepts, published online ahead of print on 27 December 2006 J. Virol. doi:10.1128/JVI.01564-06 Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Cytomegalovirus primary envelopment occurs at large
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infoldings of the inner nuclear membrane
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Cytomegalovirus nuclear membrane infoldings
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Christopher Buser1,2, Paul Walther1, Thomas Mertens2*, Detlef Michel2
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Universitätsklinikum Ulm
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Albert-Einstein-Allee 11, 89081 Ulm, Germany.
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Corresponding author:
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Prof. Dr. Thomas Mertens
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Universitätsklinikum Ulm
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Institut für Virologie
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Albert-Einstein-Allee 11
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89081 Ulm
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Germany
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Phone: 0049 (0) 731 500 65100
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Fax: 0049 (0) 731 500 65102
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Email: [email protected]
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Word count Abstract: 99
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Word count Text: 2.166
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Figures: 5
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Tables: 1
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Zentrale Einrichtung Elektronenmikroskopie, Universität Ulm, 2Institut für Virologie,
JVI01564-06 Vers 3 Abstract
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We have investigated the morphogenesis of human and murine cytomegalovirus by
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transmission electron microscopy after high-pressure freezing, freeze substitution
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and plastic embedding. We could observe large tubular infoldings of the inner
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nuclear membrane which were free of lamina and active in primary envelopment
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and subsequent transport of capsids to the nuclear periphery. Semi-quantitative
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determination of the enlarged inner nuclear membrane area and the location of
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primary envelopment of nucleocapsids demonstrated that this structure represents a
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virus induced specialized membrane domain at which the particles are
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preferentially enveloped. This is a previously not described structural element
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relevant in cytomegalovirus morphogenesis.
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JVI01564-06 Vers 3 Murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) are members of
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the Betaherpesvirinae. Both viruses encode for more than 200 open reading frames
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(ORFs) (17). The morphogenesis of herpesviruses is a complex process involving
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multiple interactions between viral and cellular components, especially membranes, and
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thus is of high interest for both virology and cell biology. The step-wise assembly of the
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virion has been studied extensively in alphaherpesviruses (11, 12, 20, 22). However,
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much less is known about the morphogenesis of cytomegaloviruses (CMVs) (3, 13).
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When comparing the findings from alphaherpesviruses with cytomegaloviruses it has to
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be kept in mind that the sequence homology is only partial and that the latter have a
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larger coding capacity. Since the size of all herpesvirus capsids prevents their transport
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into the cytoplasm through the nuclear pore complex, nuclear egress requires penetration
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of the nuclear membranes and the nuclear lamina, probably through an envelopment/de-
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envelopment process, which is still under debate (12, 26). This may also be different for
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CMVs, since the involved alphaherpesviral kinase US3 is not conserved in
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betaherpesviruses (8, 18). For MCMV it has been shown that the viral protein M50
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inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53,
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to form the putative capsid docking site (15). M50 then recruits cellular protein kinases
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for phosphorylation and dissolution of the nuclear lamina. Additionally, it has been
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shown for HCMV that pUL97 in concert with the cellular p32 acts by redistribution of
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lamina components (10). While the list of viral proteins involved in this process is
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growing, little ultrastructural information on nuclear egress is available. After release to
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the cytoplasm CMV capsids are tegumented and enveloped at Golgi-derived cisternae (7)
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by a wrapping process and released by fusion with the plasma membrane.
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JVI01564-06 Vers 3 We have investigated cytomegalovirus nuclear egress by electron microscopy using high-
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pressure freezing, freeze-substitution and plastic embedding. Fibroblast cell monolayers
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(3T3 murine fibroblasts for MCMV or human foreskin fibroblasts for HCMV) were
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grown on carbon coated sapphire discs (3 mm diameter; Engineering Office M.
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Wohlwend GmbH, Switzerland) and infected with an MOI of 0.5 PFU/cell (MCMV
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Smith strain or HCMV AD169). High-pressure freezing was performed 2 d post infection
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(p.i.) for MCMV and 3-4 d p.i. for HCMV with the new compact high pressure freezing
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apparatus HPF 01 (Engineering Office M. Wohlwend GmbH, Switzerland). For this the
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sapphire disc was immersed in 1-hexadecene and protected with 2 aluminium planchettes
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(23). Hexadecene is not miscible with water and thus does not serve as cryoprotectant,
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but is needed for optimal transfer of pressure and cooling to the sample. Freeze-
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substitution was done in acetone containing 1.6 % (w/v) osmium tetroxide, 0.1 % (w/v)
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uranyl acetate and 5 % (v/v) water (25) by slowly warming the samples from -90 °C to 0
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°C over a period of 18 h with a specially designed computer-controlled substitution
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apparatus (A. Ziegler and W. Fritz, Z.E. Elektronenmikroskopie, University of Ulm,
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unpublished). The samples were then kept at 0 °C and at room temperature for 30 min,
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washed with acetone and embedded in a two step Epon series (Fluka, Germany) of 50 %
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Epon in acteone for 1 h and 100 % Epon for 6 h. The Epon was polymerized for 3 d at 60
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°C. Thin sections (approximately 60-80 nm) were cut with a Leica Ultracut UCT ultra
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microtome using a diamond knife (Diatome, Switzerland), collected on bare copper grids.
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The samples were imaged with a Zeiss EM 10 or a Philips 400 transmission electron
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microscope at an acceleration voltage of 80 kV. The stereological determination of the
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surface ratio of infoldings vs. peripheral inner nuclear membrane was performed on
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MCMV-infected 3T3 fibroblasts by intersection counting using a square lattice grid of >4
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infected nuclei on >5 thin sections in 3 experiments each (4, 9). The budding events were
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counted in the same samples.
4 In both MCMV- and HCMV-infected cells we could observe membranes inside the
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nuclei which were associated with nucleocapsids and active in their primary envelopment
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(Fig. 1 and 4A, respectively). In MCMV infected cells various budding intermediates of
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capsids were visible, including fully enveloped lumenal particles (Fig. 1A and B). In thin
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sections these membranes were frequently connected with the inner nuclear membrane
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(Fig. 2A and B). These membranes represent tubular infoldings of the inner nuclear
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membrane and their lumen is continuous with the perinuclear space. The infoldings
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possessed a complex tubular and sometimes branched structure with total lengths of up to
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3 µm and variable diameters up to 400 nm (Fig. 2A). In contrast to the unmodified inner
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nuclear membrane they appear free of lamina (Fig. 2B, dotted lines), which is essential
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for their accessibility by the nucleocapsids in order to allow budding. In contrast to
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previous publications on pseudorabies virus morphogenesis (12), we very rarely found
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budding intermediates at the nuclear periphery, but almost exclusively at the infoldings
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(Fig. 1A). To exclude the possibility that this observation was based on an unspecific
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effect due to the enlargement of the inner nuclear membrane area, a stereological
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estimation of the surface ratio of infolded vs. peripheral inner nuclear membrane was
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performed and combined with a statistic of the location of the primary envelopment
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(Table 1). The intersection counting on infected nuclei revealed that the infolded inner
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nuclear membrane represented 4.8 % (n = 1347) of the total inner nuclear membrane
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JVI01564-06 Vers 3 area, while 86 % (n = 122) of the nucleocapsid budding profiles were located to it.
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Additionally, 93 % (n = 133) of the primary enveloped capsids were observed in the
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lumen of the infoldings compared to the peripheral perinuclear space, which suggests an
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immediate fusion with the outer nuclear membrane at the stem of the infoldings.
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Interestingly, this massive structural alteration of the inner nuclear membrane had no
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visible effect on the integrity or shape of the outer nuclear membrane or the nuclear shape
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in general. Also the nuclear pores appeared normal and not disrupted as published for
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bovine herpesvirus-1 (26). In this context it was surprising that in our samples we never
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found primary enveloped virions in the process of fusion with the outer nuclear
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membrane, only cytoplasmic capsids in close proximity to the nucleus (Fig. 3A).
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Comparison of HCMV (AD169) and MCMV nuclear egress in the two fibroblast systems
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showed some differences in appearance in spite of the high genomic sequence similarity
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of the two viruses. We observed that electron dense material between the capsids and the
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primary envelope, the putative primary tegument, is better visible in HCMV than in
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MCMV virions (Fig. 4A). Additionally, in MCMV infected 3T3 the budding at the
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infolded inner nuclear membrane was exclusively initiated by C-capsids (Fig. 1 and 2A),
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while in HCMV infected HFF also enveloped B-capsids could be observed in the lumen
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of the infoldings (Fig. 4A). Accordingly, in the cytoplasm of the HCMV infected
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fibroblasts a significant amount of tegumented and fully enveloped B-capsids was
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observed, while in MCMV infected cells capsids outside the nucleus were almost
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exclusively loaded with DNA (Fig. 3B). Additionally, MCMV did not form any dense
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bodies (Fig. 3B) in contrast to the high number seen in HCMV AD169 infected cells
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(Fig. 4B).
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JVI01564-06 Vers 3 1 Our knowledge of cytomegalovirus morphogenesis and release from the infected cell is
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still incomplete. Most of the research on general herpesviral morphogenesis has been
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done on alphaherpesviruses, e.g. herpes simplex virus and pseudorabies virus. While
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these studies have greatly enhanced our understanding of the general features of this
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process, their results should be applied to cytomegaloviruses with caution, since the
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sequence homology is only partial and the coding capacity of cytomegaloviruses is
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significantly larger. Interestingly, the homology of the proteins involved in the initial
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steps of nuclear egress is relatively high but decreases for later events. In secondary
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tegumentation and envelopment very few homologies are known, e.g. in the innermost
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tegument and the glycoproteins (11). Most models of herpesvirus morphogenesis rely on
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TEM data acquired by chemical fixation of cell cultures that has been shown to induce
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several artifacts (2, 16, 24). In recent years cryo-fixation methods have been established
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for routine use and now represent a state of the art of biological specimen preparation for
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electron microscopy (6). With cryo-fixation by high-pressure freezing, a sample, such as
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infected cultivated cells, can be fixed from a defined physiological state within
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milliseconds (14). In this study we used high-pressure freezing followed by freeze
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substitution and plastic embedding, which allows us to obtain ultra-thin sections of cryo-
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fixed samples that are almost not beam sensitive and reveal high contrast of membranous
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structures (25). An even more native state could be achieved by directly imaging the
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frozen samples in the cryo-TEM (reviewed by (1)). With this method, however, native
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cryo-sectioning of adherent cells would be very difficult and in addition, cryo-sections
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are very sensitive to beam damage, so that the samples have to be imaged at low-dose
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conditions, which decreases the signal to noise ratio.
3 We were able to preserve large invaginations of the inner nuclear membrane reaching
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lengths of few micrometers and diameters of up to several hundreds of nanometers (Fig.
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2A and B). This additional intranuclear membrane surface was highly and specifically
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involved in primary envelopment of CMV nucleocapsids as estimated by statistical
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analysis of MCMV infected cells (Table 1). Smith and de Harven, 1973 (21) also showed
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modifications of the nuclear membranes connected to nuclear egress on chemically fixed
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samples, but these were not regarded as a crucial step in the morphogenesis. Also in
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human herpesvirus-6 infected cells, a further modification of both nuclear membranes
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was observed and termed tegusome (19). The main differences between our infoldings
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and the tegusomes are that the latter are limited by a double membrane and their lumen is
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equivalent to the cytoplasm, while the infoldings described here are single-membraned
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and the lumen is continuous with the perinuclear space.
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In contrast to previous reports and the widely accepted pathway of herpesvirus nuclear
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egress we rarely found capsids budding at the nuclear periphery but instead very
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frequently at the infoldings. It has been reported that in MCMV cellular kinases are
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punctually recruited to the inner nuclear membrane by a complex of M50/M53, thus
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triggering the depolymerisation of the nuclear lamina (15). We propose that this process
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is not responsible for the formation of individual budding sites for nucleocapsids but
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instead allows the formation of the observed membrane infoldings that allow CVM
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JVI01564-06 Vers 3 primary envelopment (Fig. 5). This hypothesis is supported by the observation that the
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reported recruitment of kinases occurs at approximately 10-15 points inside the nucleus
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(15), which is comparable with the frequency of invaginations we have observed in the
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TEM (Fig. 1A). The proposed mechanism has advantages over a budding at the nuclear
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periphery. Firstly, the formation of the infoldings leads to an increase of inner nuclear
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membrane surface area which is free of lamina and thus is fully accessible for
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nucleocapsids to undergo primary envelopment (Fig. 1). Secondly, inside the tubular
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infolding the transport is directed along the tubule, since it is continuous with the
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perinuclear space, and not hindered by obstacles (e.g. chromatin). It can be expected that
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this mechanism enhances the efficiency of the transport of the primary enveloped capsids
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to the nuclear periphery, where they probably fuse with the outer nuclear membrane.
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Thirdly, the observation that the density of the nuclear lamina appears only reduced at the
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stem of the infoldings while it is still visible at the nuclear periphery (Fig. 2B, dotted
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lines) has the consequence that the outer nuclear membrane is structurally unaffected and
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the overall stability of the nucleus is retained. Interestingly, we never found primary
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enveloped virions in the process of fusion with the outer nuclear membrane. There are
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two possible explanations for this phenomenon: i) Fusion intermediates are very short-
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lived stages and more than one order of magnitude faster than the budding process. In this
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case at a given time point the total number of fusion profiles is very low and thus it is
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very improbable to observe such a profile in a thin section (60 – 80 nm thickness).
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Additionally, if fusion occurs within a few milliseconds, it might even be impossible to
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retain it, since it would be in the range of the immobilization time of the freezing process.
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ii) Primary enveloped nucleocapsids do not fuse with the outer nuclear membrane, but are
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transported along the secretory pathway. This would imply that naked cytoplasmic
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capsids arise by passage through disrupted nuclear pores as proposed by Wild et al. (26).
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We favor the first explanation because primary enveloped CMVs (Fig. 1B and 4A)
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possess a less dense tegument than mature virions and the vast majority of the enveloped
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virions in the cytoplasm are heavily tegumented as expected for secondary tegumentation
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and envelopment.
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In summary, the advantages of high-pressure freezing followed by freeze-substitution
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and plastic embedding are optimal for the visualization of short-lived and labile
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membrane structures in virus morphogenesis (5). As shown previously, the addition of 5
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% of water to the substitution medium enhances the visibility and retention of the cellular
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morphology, especially of membranes (25).
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Acknowledgements
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TM
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Kompetenznetzwerk “Resistenzentwicklung humanpathogener Erreger” TP12.
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PW and TM are supported by the Deutsche Forschungsgemeinschaft within the
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Schwerpunktprogramm SPP 1175.
and
DM
are
supported
by
the
Landesstiftung
Baden-Württemberg,
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Table Legends
2 Table 1: Statistical analysis of MCMV primary envelopment at infolded inner
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nuclear membranes. The budding profiles at the infolded vs. peripheral inner nuclear
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membranes and the location of primary enveloped virions were counted in MCMV
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infected 3T3 fibroblasts (absolute numbers). For direct correlation, the ratio of infolded
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vs. peripheral inner nuclear membrane surface was estimated on the same samples by
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counting the intersections of the inner nuclear membranes with a square lattice grid
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(absolute numbers). Connecting these two statistics shows that 86 % of nucleocapsids
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bud at infoldings, which only constitute 4.8 % of the total inner nuclear membrane
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surface. In agreement with our model that the fusion with the outer nuclear membrane
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occurs immediately at the stem of the infoldings we also found 93 % of the primary
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enveloped virions in the lumen of infoldings and only 7 % in the peripheral perinuclear
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space. INM, inner nuclear membrane; PS perinuclear space.
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Tables
budding events on
intersections with
INM infoldings
peripheral INM
INM infoldings
peripheral INM
105
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64
1283
124
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86%
14%
4.8 %
95.2 %
93%
7%
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primary enveloped virions in
Table 1
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INM infoldings peripheral PS
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Figure Legends
2 Figure 1: Infoldings of the inner nuclear membrane in MCMV infected 3T3-
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fibroblasts 50 h post infection. (A) The overview image of an infected nucleus shows
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several cross-sectioned infoldings with associated capsids. Note that only C-capsids are
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observed to undergo primary envelopment in contrast to HCMV (Fig. 4A) (bar 1 µm).
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(B) Magnified cross-section through an infolding with all intermediate stages of primary
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envelopment visible (bar 100 nm). Cy, cytoplasm; Nu, nucleus; PS, perinuclear space.
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Figure 2: Continuity of the infoldings with the inner nuclear membrane in MCMV
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infected 3T3-fibroblasts 50 h post infection. (A) Longitudinal sections through the
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infoldings show the connectivity with the inner nuclear membrane and that the apparently
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detached vesicular profiles represent complex tubules connected out of section
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(arrowheads). As quantified in Table 1, most nucleocapsids undergo primary
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envelopment at the infolded inner nuclear membrane with few exceptions that appear to
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bud at the nuclear periphery (arrows) (bar 500 nm). (B) At higher magnifications,
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longitudinally sectioned infoldings clearly show the connection (arrow) with the
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perinuclear space (PS) and the lack of lamina on the infolding (compare the areas at the
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nuclear periphery and the infolding framed by the dotted lines). Again, the stem of the
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infolding is narrow and apparently constricted by the nuclear lamina. Additionally, two
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budding capsids can be seen (bar 200 nm). Nu, nucleus; Cy, cytoplasm; PS, perinuclear
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space.
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Figure 3: Cytoplasm of 3T3 fibroblasts infected with MCMV at 50 h p.i. (A)
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Cytoplasmic capsids in close proximity to the outer nuclear membrane probably newly
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released from the perinuclear space (bar 200 nm). (B) Overview of the cytoplasm of an
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infected cell showing cytoplasmic capsid clusters typical for MCMV (arrowhead) and
5
secondary envelopment events in the area of the Golgi-complex (arrows). Note that no
6
dense bodies are present (bar 500 nm). Nu, nucleus; Cy, cytoplasm; E, late endosomes;
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G, Golgi-complex and trans-Golgi network; M, mitochondria.
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Figure 4: HCMV morphogenesis in human fibroblast cells at 4 d p.i. (A) Cross-
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section through an infolded inner nuclear membrane of a fibroblast cell infected with
11
HCMV AD169. The inner nuclear membrane is complexly folded and harbours primary
12
enveloped C- and B-capsids in which the putative primary tegument is well visible
13
(arrow; bar 200 nm). (B) Overview of the cytoplasm of an infected cell showing two
14
cross-sectioned infoldings of the inner nuclear membrane (In), cytoplasmic events of
15
dense body formation (arrows) and an enveloped cisternal virion (arrowhead). Note the
16
large amount of dense bodies (bar 1 µm). Nu, nucleus; Cy, cytoplasm; G, Golgi-complex
17
and trans-Golgi network; M, mitochondria..
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Figure 5: Model of cytomegalovirus nuclear egress. MCMV and HCMV
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morphogenesis proceeds by loading of B-capsids with viral DNA to yield the mature C-
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capsid. In parallel, the nuclear lamina is locally dissolved to generate the delaminated
22
infoldings of the inner nuclear membrane, which are forming long intranuclear tubular
23
cisterns. Nucleocapsids then make contact to these membranes over a putative primary
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tegument to undergo primary envelopment. The primary enveloped virions are
2
transported along the tubule to the nuclear periphery and probably fuse to the outer
3
nuclear membrane in a fast process, releasing the naked capsids to the cytoplasm. There,
4
the capsids acquire the two layers of secondary tegument and undergo wrapping by
5
cisternae in the area of the Golgi-complex, followed by secretion of the mature virion
6
from the cell. Nu, nucleus; Cy, cytoplasm; PS, perinuclear space; G, Golgi-complex; La,
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nuclear lamina; TGN, trans-Golgi network.
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Cytomegalovirus primary envelopment occurs at large
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infoldings of the inner nuclear membrane
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Cytomegalovirus nuclear membrane infoldings
4
Christopher Buser1,2, Paul Walther1, Thomas Mertens2*, Detlef Michel2
5
1
Universitätsklinikum Ulm
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Albert-Einstein-Allee 11, 89081 Ulm, Germany.
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Corresponding author:
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Prof. Dr. Thomas Mertens
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Universitätsklinikum Ulm
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Institut für Virologie
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Albert-Einstein-Allee 11
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89081 Ulm
15
Germany
16
Phone: 0049 (0) 731 500 65100
17
Fax: 0049 (0) 731 500 65102
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Email: [email protected]
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Word count Abstract: 99
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Word count Text: 2.166
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Figures: 5
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Tables: 1
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Zentrale Einrichtung Elektronenmikroskopie, Universität Ulm, 2Institut für Virologie,
JVI01564-06 Vers 3 Abstract
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We have investigated the morphogenesis of human and murine cytomegalovirus by
3
transmission electron microscopy after high-pressure freezing, freeze substitution
4
and plastic embedding. We could observe large tubular infoldings of the inner
5
nuclear membrane which were free of lamina and active in primary envelopment
6
and subsequent transport of capsids to the nuclear periphery. Semi-quantitative
7
determination of the enlarged inner nuclear membrane area and the location of
8
primary envelopment of nucleocapsids demonstrated that this structure represents a
9
virus induced specialized membrane domain at which the particles are
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preferentially enveloped. This is a previously not described structural element
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relevant in cytomegalovirus morphogenesis.
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JVI01564-06 Vers 3 Murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) are members of
2
the Betaherpesvirinae. Both viruses encode for more than 200 open reading frames
3
(ORFs) (17). The morphogenesis of herpesviruses is a complex process involving
4
multiple interactions between viral and cellular components, especially membranes, and
5
thus is of high interest for both virology and cell biology. The step-wise assembly of the
6
virion has been studied extensively in alphaherpesviruses (11, 12, 20, 22). However,
7
much less is known about the morphogenesis of cytomegaloviruses (CMVs) (3, 13).
8
When comparing the findings from alphaherpesviruses with cytomegaloviruses it has to
9
be kept in mind that the sequence homology is only partial and that the latter have a
10
larger coding capacity. Since the size of all herpesvirus capsids prevents their transport
11
into the cytoplasm through the nuclear pore complex, nuclear egress requires penetration
12
of the nuclear membranes and the nuclear lamina, probably through an envelopment/de-
13
envelopment process, which is still under debate (12, 26). This may also be different for
14
CMVs, since the involved alphaherpesviral kinase US3 is not conserved in
15
betaherpesviruses (8, 18). For MCMV it has been shown that the viral protein M50
16
inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53,
17
to form the putative capsid docking site (15). M50 then recruits cellular protein kinases
18
for phosphorylation and dissolution of the nuclear lamina. Additionally, it has been
19
shown for HCMV that pUL97 in concert with the cellular p32 acts by redistribution of
20
lamina components (10). While the list of viral proteins involved in this process is
21
growing, little ultrastructural information on nuclear egress is available. After release to
22
the cytoplasm CMV capsids are tegumented and enveloped at Golgi-derived cisternae (7)
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by a wrapping process and released by fusion with the plasma membrane.
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JVI01564-06 Vers 3 We have investigated cytomegalovirus nuclear egress by electron microscopy using high-
2
pressure freezing, freeze-substitution and plastic embedding. Fibroblast cell monolayers
3
(3T3 murine fibroblasts for MCMV or human foreskin fibroblasts for HCMV) were
4
grown on carbon coated sapphire discs (3 mm diameter; Engineering Office M.
5
Wohlwend GmbH, Switzerland) and infected with an MOI of 0.5 PFU/cell (MCMV
6
Smith strain or HCMV AD169). High-pressure freezing was performed 2 d post infection
7
(p.i.) for MCMV and 3-4 d p.i. for HCMV with the new compact high pressure freezing
8
apparatus HPF 01 (Engineering Office M. Wohlwend GmbH, Switzerland). For this the
9
sapphire disc was immersed in 1-hexadecene and protected with 2 aluminium planchettes
10
(23). Hexadecene is not miscible with water and thus does not serve as cryoprotectant,
11
but is needed for optimal transfer of pressure and cooling to the sample. Freeze-
12
substitution was done in acetone containing 1.6 % (w/v) osmium tetroxide, 0.1 % (w/v)
13
uranyl acetate and 5 % (v/v) water (25) by slowly warming the samples from -90 °C to 0
14
°C over a period of 18 h with a specially designed computer-controlled substitution
15
apparatus (A. Ziegler and W. Fritz, Z.E. Elektronenmikroskopie, University of Ulm,
16
unpublished). The samples were then kept at 0 °C and at room temperature for 30 min,
17
washed with acetone and embedded in a two step Epon series (Fluka, Germany) of 50 %
18
Epon in acteone for 1 h and 100 % Epon for 6 h. The Epon was polymerized for 3 d at 60
19
°C. Thin sections (approximately 60-80 nm) were cut with a Leica Ultracut UCT ultra
20
microtome using a diamond knife (Diatome, Switzerland), collected on bare copper grids.
21
The samples were imaged with a Zeiss EM 10 or a Philips 400 transmission electron
22
microscope at an acceleration voltage of 80 kV. The stereological determination of the
23
surface ratio of infoldings vs. peripheral inner nuclear membrane was performed on
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MCMV-infected 3T3 fibroblasts by intersection counting using a square lattice grid of >4
2
infected nuclei on >5 thin sections in 3 experiments each (4, 9). The budding events were
3
counted in the same samples.
4 In both MCMV- and HCMV-infected cells we could observe membranes inside the
6
nuclei which were associated with nucleocapsids and active in their primary envelopment
7
(Fig. 1 and 4A, respectively). In MCMV infected cells various budding intermediates of
8
capsids were visible, including fully enveloped lumenal particles (Fig. 1A and B). In thin
9
sections these membranes were frequently connected with the inner nuclear membrane
10
(Fig. 2A and B). These membranes represent tubular infoldings of the inner nuclear
11
membrane and their lumen is continuous with the perinuclear space. The infoldings
12
possessed a complex tubular and sometimes branched structure with total lengths of up to
13
3 µm and variable diameters up to 400 nm (Fig. 2A). In contrast to the unmodified inner
14
nuclear membrane they appear free of lamina (Fig. 2B, dotted lines), which is essential
15
for their accessibility by the nucleocapsids in order to allow budding. In contrast to
16
previous publications on pseudorabies virus morphogenesis (12), we very rarely found
17
budding intermediates at the nuclear periphery, but almost exclusively at the infoldings
18
(Fig. 1A). To exclude the possibility that this observation was based on an unspecific
19
effect due to the enlargement of the inner nuclear membrane area, a stereological
20
estimation of the surface ratio of infolded vs. peripheral inner nuclear membrane was
21
performed and combined with a statistic of the location of the primary envelopment
22
(Table 1). The intersection counting on infected nuclei revealed that the infolded inner
23
nuclear membrane represented 4.8 % (n = 1347) of the total inner nuclear membrane
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Additionally, 93 % (n = 133) of the primary enveloped capsids were observed in the
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lumen of the infoldings compared to the peripheral perinuclear space, which suggests an
4
immediate fusion with the outer nuclear membrane at the stem of the infoldings.
5
Interestingly, this massive structural alteration of the inner nuclear membrane had no
6
visible effect on the integrity or shape of the outer nuclear membrane or the nuclear shape
7
in general. Also the nuclear pores appeared normal and not disrupted as published for
8
bovine herpesvirus-1 (26). In this context it was surprising that in our samples we never
9
found primary enveloped virions in the process of fusion with the outer nuclear
10
membrane, only cytoplasmic capsids in close proximity to the nucleus (Fig. 3A).
11
Comparison of HCMV (AD169) and MCMV nuclear egress in the two fibroblast systems
12
showed some differences in appearance in spite of the high genomic sequence similarity
13
of the two viruses. We observed that electron dense material between the capsids and the
14
primary envelope, the putative primary tegument, is better visible in HCMV than in
15
MCMV virions (Fig. 4A). Additionally, in MCMV infected 3T3 the budding at the
16
infolded inner nuclear membrane was exclusively initiated by C-capsids (Fig. 1 and 2A),
17
while in HCMV infected HFF also enveloped B-capsids could be observed in the lumen
18
of the infoldings (Fig. 4A). Accordingly, in the cytoplasm of the HCMV infected
19
fibroblasts a significant amount of tegumented and fully enveloped B-capsids was
20
observed, while in MCMV infected cells capsids outside the nucleus were almost
21
exclusively loaded with DNA (Fig. 3B). Additionally, MCMV did not form any dense
22
bodies (Fig. 3B) in contrast to the high number seen in HCMV AD169 infected cells
23
(Fig. 4B).
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JVI01564-06 Vers 3 1 Our knowledge of cytomegalovirus morphogenesis and release from the infected cell is
3
still incomplete. Most of the research on general herpesviral morphogenesis has been
4
done on alphaherpesviruses, e.g. herpes simplex virus and pseudorabies virus. While
5
these studies have greatly enhanced our understanding of the general features of this
6
process, their results should be applied to cytomegaloviruses with caution, since the
7
sequence homology is only partial and the coding capacity of cytomegaloviruses is
8
significantly larger. Interestingly, the homology of the proteins involved in the initial
9
steps of nuclear egress is relatively high but decreases for later events. In secondary
10
tegumentation and envelopment very few homologies are known, e.g. in the innermost
11
tegument and the glycoproteins (11). Most models of herpesvirus morphogenesis rely on
12
TEM data acquired by chemical fixation of cell cultures that has been shown to induce
13
several artifacts (2, 16, 24). In recent years cryo-fixation methods have been established
14
for routine use and now represent a state of the art of biological specimen preparation for
15
electron microscopy (6). With cryo-fixation by high-pressure freezing, a sample, such as
16
infected cultivated cells, can be fixed from a defined physiological state within
17
milliseconds (14). In this study we used high-pressure freezing followed by freeze
18
substitution and plastic embedding, which allows us to obtain ultra-thin sections of cryo-
19
fixed samples that are almost not beam sensitive and reveal high contrast of membranous
20
structures (25). An even more native state could be achieved by directly imaging the
21
frozen samples in the cryo-TEM (reviewed by (1)). With this method, however, native
22
cryo-sectioning of adherent cells would be very difficult and in addition, cryo-sections
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are very sensitive to beam damage, so that the samples have to be imaged at low-dose
2
conditions, which decreases the signal to noise ratio.
3 We were able to preserve large invaginations of the inner nuclear membrane reaching
5
lengths of few micrometers and diameters of up to several hundreds of nanometers (Fig.
6
2A and B). This additional intranuclear membrane surface was highly and specifically
7
involved in primary envelopment of CMV nucleocapsids as estimated by statistical
8
analysis of MCMV infected cells (Table 1). Smith and de Harven, 1973 (21) also showed
9
modifications of the nuclear membranes connected to nuclear egress on chemically fixed
10
samples, but these were not regarded as a crucial step in the morphogenesis. Also in
11
human herpesvirus-6 infected cells, a further modification of both nuclear membranes
12
was observed and termed tegusome (19). The main differences between our infoldings
13
and the tegusomes are that the latter are limited by a double membrane and their lumen is
14
equivalent to the cytoplasm, while the infoldings described here are single-membraned
15
and the lumen is continuous with the perinuclear space.
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In contrast to previous reports and the widely accepted pathway of herpesvirus nuclear
18
egress we rarely found capsids budding at the nuclear periphery but instead very
19
frequently at the infoldings. It has been reported that in MCMV cellular kinases are
20
punctually recruited to the inner nuclear membrane by a complex of M50/M53, thus
21
triggering the depolymerisation of the nuclear lamina (15). We propose that this process
22
is not responsible for the formation of individual budding sites for nucleocapsids but
23
instead allows the formation of the observed membrane infoldings that allow CVM
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JVI01564-06 Vers 3 primary envelopment (Fig. 5). This hypothesis is supported by the observation that the
2
reported recruitment of kinases occurs at approximately 10-15 points inside the nucleus
3
(15), which is comparable with the frequency of invaginations we have observed in the
4
TEM (Fig. 1A). The proposed mechanism has advantages over a budding at the nuclear
5
periphery. Firstly, the formation of the infoldings leads to an increase of inner nuclear
6
membrane surface area which is free of lamina and thus is fully accessible for
7
nucleocapsids to undergo primary envelopment (Fig. 1). Secondly, inside the tubular
8
infolding the transport is directed along the tubule, since it is continuous with the
9
perinuclear space, and not hindered by obstacles (e.g. chromatin). It can be expected that
10
this mechanism enhances the efficiency of the transport of the primary enveloped capsids
11
to the nuclear periphery, where they probably fuse with the outer nuclear membrane.
12
Thirdly, the observation that the density of the nuclear lamina appears only reduced at the
13
stem of the infoldings while it is still visible at the nuclear periphery (Fig. 2B, dotted
14
lines) has the consequence that the outer nuclear membrane is structurally unaffected and
15
the overall stability of the nucleus is retained. Interestingly, we never found primary
16
enveloped virions in the process of fusion with the outer nuclear membrane. There are
17
two possible explanations for this phenomenon: i) Fusion intermediates are very short-
18
lived stages and more than one order of magnitude faster than the budding process. In this
19
case at a given time point the total number of fusion profiles is very low and thus it is
20
very improbable to observe such a profile in a thin section (60 – 80 nm thickness).
21
Additionally, if fusion occurs within a few milliseconds, it might even be impossible to
22
retain it, since it would be in the range of the immobilization time of the freezing process.
23
ii) Primary enveloped nucleocapsids do not fuse with the outer nuclear membrane, but are
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transported along the secretory pathway. This would imply that naked cytoplasmic
2
capsids arise by passage through disrupted nuclear pores as proposed by Wild et al. (26).
3
We favor the first explanation because primary enveloped CMVs (Fig. 1B and 4A)
4
possess a less dense tegument than mature virions and the vast majority of the enveloped
5
virions in the cytoplasm are heavily tegumented as expected for secondary tegumentation
6
and envelopment.
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In summary, the advantages of high-pressure freezing followed by freeze-substitution
9
and plastic embedding are optimal for the visualization of short-lived and labile
10
membrane structures in virus morphogenesis (5). As shown previously, the addition of 5
11
% of water to the substitution medium enhances the visibility and retention of the cellular
12
morphology, especially of membranes (25).
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Acknowledgements
2
TM
3
Kompetenznetzwerk “Resistenzentwicklung humanpathogener Erreger” TP12.
4
PW and TM are supported by the Deutsche Forschungsgemeinschaft within the
5
Schwerpunktprogramm SPP 1175.
and
DM
are
supported
by
the
Landesstiftung
Baden-Württemberg,
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Table Legends
2 Table 1: Statistical analysis of MCMV primary envelopment at infolded inner
4
nuclear membranes. The budding profiles at the infolded vs. peripheral inner nuclear
5
membranes and the location of primary enveloped virions were counted in MCMV
6
infected 3T3 fibroblasts (absolute numbers). For direct correlation, the ratio of infolded
7
vs. peripheral inner nuclear membrane surface was estimated on the same samples by
8
counting the intersections of the inner nuclear membranes with a square lattice grid
9
(absolute numbers). Connecting these two statistics shows that 86 % of nucleocapsids
10
bud at infoldings, which only constitute 4.8 % of the total inner nuclear membrane
11
surface. In agreement with our model that the fusion with the outer nuclear membrane
12
occurs immediately at the stem of the infoldings we also found 93 % of the primary
13
enveloped virions in the lumen of infoldings and only 7 % in the peripheral perinuclear
14
space. INM, inner nuclear membrane; PS perinuclear space.
15 16 17
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Tables
budding events on
intersections with
INM infoldings
peripheral INM
INM infoldings
peripheral INM
105
17
64
1283
124
9
86%
14%
4.8 %
95.2 %
93%
7%
18 19
primary enveloped virions in
Table 1
16
INM infoldings peripheral PS
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Figure Legends
2 Figure 1: Infoldings of the inner nuclear membrane in MCMV infected 3T3-
4
fibroblasts 50 h post infection. (A) The overview image of an infected nucleus shows
5
several cross-sectioned infoldings with associated capsids. Note that only C-capsids are
6
observed to undergo primary envelopment in contrast to HCMV (Fig. 4A) (bar 1 µm).
7
(B) Magnified cross-section through an infolding with all intermediate stages of primary
8
envelopment visible (bar 100 nm). Cy, cytoplasm; Nu, nucleus; PS, perinuclear space.
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T P
9
E C
10
Figure 2: Continuity of the infoldings with the inner nuclear membrane in MCMV
11
infected 3T3-fibroblasts 50 h post infection. (A) Longitudinal sections through the
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infoldings show the connectivity with the inner nuclear membrane and that the apparently
13
detached vesicular profiles represent complex tubules connected out of section
14
(arrowheads). As quantified in Table 1, most nucleocapsids undergo primary
15
envelopment at the infolded inner nuclear membrane with few exceptions that appear to
16
bud at the nuclear periphery (arrows) (bar 500 nm). (B) At higher magnifications,
17
longitudinally sectioned infoldings clearly show the connection (arrow) with the
18
perinuclear space (PS) and the lack of lamina on the infolding (compare the areas at the
19
nuclear periphery and the infolding framed by the dotted lines). Again, the stem of the
20
infolding is narrow and apparently constricted by the nuclear lamina. Additionally, two
21
budding capsids can be seen (bar 200 nm). Nu, nucleus; Cy, cytoplasm; PS, perinuclear
22
space.
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Figure 3: Cytoplasm of 3T3 fibroblasts infected with MCMV at 50 h p.i. (A)
2
Cytoplasmic capsids in close proximity to the outer nuclear membrane probably newly
3
released from the perinuclear space (bar 200 nm). (B) Overview of the cytoplasm of an
4
infected cell showing cytoplasmic capsid clusters typical for MCMV (arrowhead) and
5
secondary envelopment events in the area of the Golgi-complex (arrows). Note that no
6
dense bodies are present (bar 500 nm). Nu, nucleus; Cy, cytoplasm; E, late endosomes;
7
G, Golgi-complex and trans-Golgi network; M, mitochondria.
T P
8 9
Figure 4: HCMV morphogenesis in human fibroblast cells at 4 d p.i. (A) Cross-
10
section through an infolded inner nuclear membrane of a fibroblast cell infected with
11
HCMV AD169. The inner nuclear membrane is complexly folded and harbours primary
12
enveloped C- and B-capsids in which the putative primary tegument is well visible
13
(arrow; bar 200 nm). (B) Overview of the cytoplasm of an infected cell showing two
14
cross-sectioned infoldings of the inner nuclear membrane (In), cytoplasmic events of
15
dense body formation (arrows) and an enveloped cisternal virion (arrowhead). Note the
16
large amount of dense bodies (bar 1 µm). Nu, nucleus; Cy, cytoplasm; G, Golgi-complex
17
and trans-Golgi network; M, mitochondria..
E C
C A
18 19
Figure 5: Model of cytomegalovirus nuclear egress. MCMV and HCMV
20
morphogenesis proceeds by loading of B-capsids with viral DNA to yield the mature C-
21
capsid. In parallel, the nuclear lamina is locally dissolved to generate the delaminated
22
infoldings of the inner nuclear membrane, which are forming long intranuclear tubular
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cisterns. Nucleocapsids then make contact to these membranes over a putative primary
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tegument to undergo primary envelopment. The primary enveloped virions are
2
transported along the tubule to the nuclear periphery and probably fuse to the outer
3
nuclear membrane in a fast process, releasing the naked capsids to the cytoplasm. There,
4
the capsids acquire the two layers of secondary tegument and undergo wrapping by
5
cisternae in the area of the Golgi-complex, followed by secretion of the mature virion
6
from the cell. Nu, nucleus; Cy, cytoplasm; PS, perinuclear space; G, Golgi-complex; La,
7
nuclear lamina; TGN, trans-Golgi network.
T P
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C A
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