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Bernard Moss - One of the best experts on this subject based on the ideXlab platform.
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Assembly and Disassembly of the Capsid-Like External Scaffold of Immature Virions during Vaccinia Virus Morphogenesis
Journal of virology, 2009Co-Authors: Himani Bisht, Patricia Szajner, Andrea S. Weisberg, Bernard MossAbstract:Infectious poxVirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxVirus particles. In the case of vaccinia Virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of Morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant Virus particles. Furthermore, the Morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.
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Evidence for an Essential Catalytic Role of the F10 Protein Kinase in Vaccinia Virus Morphogenesis
Journal of virology, 2004Co-Authors: Patricia Szajner, Andrea S. Weisberg, Bernard MossAbstract:Temperature-sensitive mutants of vaccinia Virus, with genetic changes that map to the open reading frame encoding the F10 protein kinase, exhibit a defect at an early stage of viral Morphogenesis. To further study the role of the enzyme, we constructed recombinant vaccinia Virus vF10V5i, which expresses inducible V5 epitope-tagged F10 and is dependent on a chemical inducer for plaque formation and replication. In the absence of inducer, viral membrane formation was delayed and crescents and occasional immature forms were detected only late in infection. When the temperature was raised from 37 to 39°C, the block in membrane formation persisted throughout the infection. The increased stringency may be explained by a mild temperature sensitivity of the wild-type F10 kinase, which reduced the activity of the very small amount expressed in the absence of inducer, or by the thermolability of an unphosphorylated kinase substrate or uncomplexed F10-interacting protein. Further analyses demonstrated that tyrosine and threonine phosphorylation of the A17 membrane component was inhibited in the absence of inducer. The phosphorylation defect could be overcome by transfection of plasmids that express wild-type F10, but not by plasmids that express F10 with single amino acid substitutions that abolished catalytic activity. Although the mutated forms of F10 were stable and concentrated in viral factories, only the wild-type protein complemented the assembly and replication defects of vF10V5i in the absence of inducer. These studies provide evidence for an essential catalytic role of the F10 kinase in vaccinia Virus Morphogenesis.
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Regulation of Vaccinia Virus Morphogenesis: Phosphorylation of the A14L and A17L Membrane Proteins and C-Terminal Truncation of the A17L Protein Are Dependent on the F10L Kinase
Journal of virology, 1999Co-Authors: Tatiana Betakova, Elizabeth J. Wolffe, Bernard MossAbstract:This study focused on three vaccinia Virus-encoded proteins that participate in early steps of virion Morphogenesis: the A17L and A14L membrane proteins and the F10L protein kinase. We found that (i) the A17L protein was cleaved at or near an AGX consensus motif at amino acid 185, thereby removing its acidic C terminus; (ii) the nontruncated form was associated with immature virions, but only the C-terminal truncated protein was present in mature virions; (iii) the nontruncated form of the A17L protein was phosphorylated on serine, threonine, and tyrosine residues, whereas the truncated form was unphosphorylated; (iv) nontruncated and truncated forms of the A17L protein existed in a complex with the A14L membrane protein; (v) C-terminal cleavage of the A17L protein and phosphorylation of the A17L and A14L proteins failed to occur in cells infected with a F10L kinase mutant at the nonpermissive temperature; and (vi) the F10L kinase was the only viral late protein that was necessary for phosphorylation of the A17L protein, whereas additional proteins were needed for C-terminal cleavage. We suggest that phosphorylation of the A17L and A14L proteins is mediated by the F10L kinase and is required to form the membranes associated with immature virions. Removal of phosphates and the A17L acidic C-terminal peptide occur during the transition to mature virions. The initial steps in vaccinia Virus Morphogenesis are poorly understood. The first viral structures are crescent-shaped membranes that appear to form de novo in specialized factory regions of the cytoplasm which are largely devoid of cellular organelles (6, 10, 24). Griffiths and coworkers (36, 42) have proposed that the viral membranes are derived from the cellular intermediate compartment by a wrapping mechanism. Regardless of their origin, the crescents develop into spherical, immature virions (IV) containing the double-stranded DNA genome and subsequently into dense, brick-shaped, infectious intracellular mature virions (IMV). Some of the IMV escape from the assembly regions and are wrapped by membrane cisternae, derived from the trans-Golgi or early endosomal network, to form the intracellular enveloped virions (IEV) (13, 15, 23, 38, 47). A subset of IEV are propelled through the cytoplasm via actin tails and form the tips of specialized microvilli that protrude from the cell surface and mediate efficient cell-to-cell Virus spread (5, 12, 14, 35, 37, 44, 55, 57). IEV without actin tails also reach the periphery (55), where they fuse with the plasma membrane to form cell-associated enveloped virions and released extracellular enveloped virions (3, 28).
Richard J. Sugrue - One of the best experts on this subject based on the ideXlab platform.
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Viperin protein expression inhibits the late stage of respiratory syncytial Virus Morphogenesis
Antiviral research, 2014Co-Authors: Muhammad Raihan Jumat, Boon Huan Tan, Tra Ngyen Huong, Laxmi Iyer Ravi, Rebecca Rongrong Stanford, Richard J. SugrueAbstract:Abstract We examined the effect of respiratory syncytial Virus (RSV) infection on viperin protein expression in the permissive HEp2 and non-permissive RAW 264.7 macrophage cell lines. In RSV-infected HEp2 cells low levels of the viperin protein was localized to the Virus-induced inclusion bodies and did not impair Virus transmission in these cells. In contrast, RSV-infected RAW 264.7 cells increased expression of the STAT1 protein occurred at between 6 and 12 h post-infection, which coincided with the appearance of P-STAT1. A relatively high level of viperin protein expression was detected in infected RAW 264.7 cells, and it was extensively localized throughout the cytoplasm of infected cells. The effect of early viperin protein expression on RSV infection in cells that are normally permissive to RSV cultivation was examined by using either transient transfected HEp2 cells or stable transfected HeLa cells that expressed the viperin protein. The early expression of viperin in HeLa cells did not prevent Virus infection, and no significant inhibitory effect on either Virus protein expression or targeting of Virus proteins to the cell surface was noted. However, while inclusion body formation was not inhibited, early viperin protein expression was associated with the inhibition of Virus filament formation and reduced cell-to-cell Virus transmission. Inhibition of Virus filament formation was also observed in HEp2 cells expressing viperin. Collectively our data suggested that viperin impaired RSV transmission by inhibiting Virus filament formation, providing a basis for its anti-Virus activity in RSV-infected cells.
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Imaging analysis of human metapneumoVirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in Virus Morphogenesis.
Virology journal, 2014Co-Authors: Muhammad Raihan Jumat, Tra Nguyen Huong, Puisan Wong, Liat Hui Loo, Boon Huan Tan, Fiona Fenwick, Geoffrey L. Toms, Richard J. SugrueAbstract:Backgound: Due to difficulties of culturing Human metapneumoVirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related Viruses. The slow rates of Virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV Morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV Morphogenesis. Methods: HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and Virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed Virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined. Results: HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the Virus Morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous Virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of Virus assembly. Conclusions: These data suggest that HMPV matures as filamentous particles and that Virus Morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV Morphogenesis and the closely related human respiratory syncytial Virus suggests that involvement of F-actin and lipid-raft microdomains in Virus Morphogenesis may be a common feature of the Pneumovirinae.
José L. Carrascosa - One of the best experts on this subject based on the ideXlab platform.
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Membrane remodelling during vaccinia Virus Morphogenesis.
Biology of the cell, 2009Co-Authors: Francisco Javier Chichón, María Josefa Rodríguez, Cristina Risco, Alberto Fraile-ramos, José-jesús Fernández, Mariano Esteban, José L. CarrascosaAbstract:Background information. VACV (vaccinia Virus) is one of the most complex Viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. Results. We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the Virus during envelope maturation. The tomograms obtained suggest that a number of independent ‘crescents’ interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the Virus [IV (immature Virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature Virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. Conclusions. The main features observed for VACV membrane maturation during Morphogenesis resemble the breakdown and reassembly of cellular endomembranes.
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two types of Virus related particles are found during transmissible gastroenteritis Virus Morphogenesis
Journal of Virology, 1998Co-Authors: Cristina Risco, Maria Muntion, Luis Enjuanes, José L. CarrascosaAbstract:The intracellular assembly of the transmissible gastroenteritis coronaVirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronaVirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.
Tetsuya Yoshida - One of the best experts on this subject based on the ideXlab platform.
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Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein
Journal of virology, 2002Co-Authors: Takemasa Sakaguchi, Tsuneo Uchiyama, Cheng Huang, Noriko Fukuhara, Katsuhiro Kiyotani, Yoshiyuki Nagai, Tetsuya YoshidaAbstract:Sendai Virus (SeV), an enveloped Virus with a single-stranded negative-sense RNA genome of 15,384 bases, belongs to the genus RespiroVirus of the family Paramyxoviridae. The Virus particle displays spherical morphology of relatively uniform size with a diameter of about 200 nm. The envelope comprises a lipid bilayer derived from the host plasma membrane and two inserted viral glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) proteins. Lining beneath the envelope are the matrix or membrane (M) proteins. The nucleocapsid represents the internal structure, which comprises the genome RNA complexed with the nucleocapsid (N) protein and polymerase consisting of the L (large) protein and the P (phospho) protein (11). There is increasing evidence suggesting that the M protein plays a key role in the assembly of paramyxoViruses and related RNA Viruses. The M protein has been suggested to be essential to cross-link the external envelope proteins and the internal nucleocapsid. It also promotes the condensation of viral glycoproteins into a patch in the plasma membrane, an immediate precursor of the envelope (16, 28). The M proteins, associating with the nucleocapsid, may then provide forces from the inside for the membrane patch to bud (3, 7, 15). Bending of membranes may also be facilitated from the outside by glycoproteins (17, 18). We previously showed that the SeV M protein expressed from plasmid was released into the culture supernatant, as was seen in the case of the M protein of vesicular stomatitis Virus (12, 22). Recently, the F protein as well as the M protein was shown to cause the budding of vesicles from cells (25). The SeV M protein as well as the F protein therefore has an intrinsic nature to be a driving force of Virus budding. The SeV M protein is 348 amino acids in length and contains five cysteine residues. Cysteines can form intrachain and interchain disulfide bonds and thereby contribute to the folding of polypeptides as well as homologous and heterologous protein-protein covalent interactions. Cysteines in various enzymes function as an active center, and those in various proteins sometimes form zinc finger motifs to bind metabolically important zinc ions (5, 13). The last function has recently been exemplified for a nonstructural protein of SeV, the V protein (8). Little is known, however, about the cysteine residues of the SeV M protein. It is unlikely that the cysteines in the M protein are intracellularly oxidized to form disulfide bonds because the M protein is localized in the cytosol, the reducing milieu. However, it is not known whether the cysteines of the M protein form disulfide bonds in Virus particles in an oxidizing extracellular environment. Nevertheless, the fact that cysteine residues are well conserved in a wide variety of paramyxoVirus M proteins suggests that the functions of the M proteins are important. We therefore focused on cysteine residues at the targets of site-directed mutagenesis and investigated the actual contribution of the M protein to SeV assembly.
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Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the
2002Co-Authors: Takemasa Sakaguchi, Tsuneo Uchiyama, Cheng Huang, Noriko Fukuhara, Katsuhiro Kiyotani, Yoshiyuki Nagai, Tetsuya YoshidaAbstract:The matrix (M) protein of Sendai Virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate Virus from cDNA, SeV M-C83S, SeV M-C106S, and SeV M-C295S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C83S and SeV M-C106S had smaller Virus particles than did the wild-type SeV, whereas SeV M-C295S had larger and heterogeneously sized particles. Furthermore, SeV M-C106S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects Virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle. Sendai Virus (SeV), an enveloped Virus with a single-stranded negative-sense RNA genome of 15,384 bases, belongs to the genus RespiroVirus of the family Paramyxoviridae. The Virus particle displays spherical morphology of relatively uniform size with a diameter of about 200 nm. The envelope comprises a lipid bilayer derived from the host plasma membrane and two inserted viral glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) proteins. Lining beneath the envelope are the matrix or membrane (M) proteins. The nucleocapsid represents the internal structure, which comprises the genome RNA complexed with the nucleocapsid (N) protein and polymerase consisting of the L (large) protein and the P (phospho) protein (11). There is increasing evidence suggesting that the M protein plays a key role in the assembly of paramyxoViruses and related RNA Viruses. The M protein has been suggested to be essential to cross-link the external envelope proteins and the internal nucleocapsid. It also promotes the condensation of viral glycoproteins into a patch in the plasma membrane, an immediate precursor of the envelope (16, 28). The M proteins, associating with the nucleocapsid, may then provide forces from the inside for the membrane patch to bud (3, 7, 15). Bending of membranes may also be facilitated from the outside by glycoproteins (17, 18). We previously showed that the SeV M protein expressed from plasmid was released into the culture supernatant, as was seen in the case of the M protein of vesicular stomatitis Virus (12, 22). Recently, the F protein as well as the M protein was shown to cause the budding of vesicles from cells (25). The SeV M protein as well as the F protein therefore has an intrinsic nature to be a driving force of Virus budding. The SeV M protein is 348 amino acids in length and contains five cysteine residues. Cysteines can form intrachain and interchain disulfide bonds and thereby contribute to the folding of polypeptides as well as homologous and heterologous proteinprotein covalent interactions. Cysteines in various enzymes function as an active center, and those in various proteins sometimes form zinc finger motifs to bind metabolically important zinc ions (5, 13). The last function has recently been exemplified for a nonstructural protein of SeV, the V protein (8). Little is known, however, about the cysteine residues of the SeV M protein. It is unlikely that the cysteines in the M protein are intracellularly oxidized to form disulfide bonds because the M protein is localized in the cytosol, the reducing milieu. However, it is not known whether the cysteines of the M protein form disulfide bonds in Virus particles in an oxidizing extracellular environment. Nevertheless, the fact that cysteine residues are well conserved in a wide variety of paramyxoVirus M proteins suggests that the functions of the M proteins are important. We therefore focused on cysteine residues at the targets of site-directed mutagenesis and investigated the actual contribution of the M protein to SeV assembly.
Cristina Risco - One of the best experts on this subject based on the ideXlab platform.
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Virus Morphogenesis in the Cell: Methods and Observations
Sub-cellular biochemistry, 2013Co-Authors: Cristina Risco, Isabel Fernández De CastroAbstract:Viruses carry out many of their activities inside cells, where they synthesise proteins that are not incorporated into viral particles. Some of these proteins trigger signals to kidnap cell organelles and factors which will form a new macro-structure, the Virus factory, that acts as a physical scaffold for viral replication and assembly. We are only beginning to envisage the extraordinary complexity of these interactions, whose characterisation is a clear experimental challenge for which we now have powerful tools. Conventional study of infection kinetics using virology, biochemistry and cell biology methods can be followed by genome-scale screening and global proteomics. These are important new technologies with which we can identify the cell factors used by Viruses at different stages in their life cycle. Light microscopy, electron microscopy and electron tomography, together with labelling methods for molecular mapping in situ, show immature viral intermediates, mature virions and recruited cell elements in their natural environment. This chapter describes how these methods are being used to understand the cell biology of viral Morphogenesis and suggests what they might achieve in the near future.
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Membrane remodelling during vaccinia Virus Morphogenesis.
Biology of the cell, 2009Co-Authors: Francisco Javier Chichón, María Josefa Rodríguez, Cristina Risco, Alberto Fraile-ramos, José-jesús Fernández, Mariano Esteban, José L. CarrascosaAbstract:Background information. VACV (vaccinia Virus) is one of the most complex Viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. Results. We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the Virus during envelope maturation. The tomograms obtained suggest that a number of independent ‘crescents’ interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the Virus [IV (immature Virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature Virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. Conclusions. The main features observed for VACV membrane maturation during Morphogenesis resemble the breakdown and reassembly of cellular endomembranes.
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two types of Virus related particles are found during transmissible gastroenteritis Virus Morphogenesis
Journal of Virology, 1998Co-Authors: Cristina Risco, Maria Muntion, Luis Enjuanes, José L. CarrascosaAbstract:The intracellular assembly of the transmissible gastroenteritis coronaVirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronaVirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.
