Aspects of the developmental morphology of California encephalitis virus in cultured vertebrae and arthropod cells and in mouse brain

An electron microscopic study was made of the replication of California encephalitis (La Crosse strain) in cultured vertebrate cells (Vero, African green monkey kidney), in a line of cultured mosquito cells (Aedes albopictus), and in brain tissue of suckling mice. Morphologically similar virus particles, approximately 95 nm in diameter, were encountered in all three systems, and a common mode of virus assembly and maturation appeared to obtain. Virus assembly was shown to occur exclusively at internal cytomembrane interfaces, the Golgi complex appearing as the initial assembly site, which site became less focal as infection progressed due to the proliferation of Golgi smooth membranes and the dilation of cisternae and vesicles. The assembly process involved viral budding into cisternal and vesicular lumina, the virion thereby acquiring its limiting membrane. The envelope of such intracellular virions exhibited a poorly defined fringe, approximately 8 nm in width, which appeared to undergo a maturational change as the virions were discharged from the cell, such that, extracellularly, virions displayed a well-developed fringe, approximately 12 nm wide—a change especially noteworthy in the case of virions in infected mouse brain.The presence was noted in infected Vero cells—and in one instance in an infected mosquito cell—of crescent-shaped segments of thickened cisternal membrane, which possibly represented an early phase of viral assembly in which nucleocapsid aligned itself in close apposition to a membrane segment preparatory to the initiation of budding.In areas of the cytoplasm adjacent to sites of viral assembly in neurons, a fine granulofibrillar matrix was frequently found, enmeshed in which were numbers of 50–60 nm spherical structures. In a low proportion of cells from infected mosquito cultures, dense granulofibrillar masses were found in the cytoplasm.In Vero cells, infection with CE virus was cytolytic, while in A. albopictus cells, no gross cytopathic effects were manifest, and persistently infected cultures developed upon subcultivation. However, less than 10% of challenged mosquito cells became productively infected and for a proportion of these, at least as determined by electron microscopy, the infection was lethal.     

Molecular mechanism of paramyxovirus budding

Components of paramyxoviruses are assembled at the plasma membrane of infected cells, and progeny viruses are formed by the budding process. Although the molecular mechanisms that drive budding (membrane curving and "pinching-off" reaction) are not well understood, the viral matrix (M) protein is thought to play a major role in the process. The M protein forms a dense layer tightly associated with the inner leaflet of the plasma membrane of infected cells. Expression of the M protein of some paramyxoviruses results in the formation and release of virus-like particles that contain the M protein; thus, in these viruses, the M protein alone can apparently trigger all steps required for the formation and release of virus-like particles. M also interacts specifically with viral envelope glycoproteins and nucleocapsids and is involved in directed transport of viral components to the budding site at the apical surface of polarized cells. In addition, protein-protein interactions between M and the cytoplasmic tail of viral glycoproteins and between M and the nucleocapsid affect the efficiency of virus production. The structural organization of the virion and the functions of the M protein clearly indicate that this protein orchestrates the budding of paramyxovirus.     

Ultrastructural-immunohistochemical evidence for a maturation defect of temperature-sensitive G31 vesicular stomatitis virus in murine spinal cord neurons.

Ultrastructural immunoperoxidase studies were done in spinal cords of mice infected with wild type vesicular stomatitis virus or its temperature-sensitive (ts) mutant G31. Infected neurons showed subplasmalemmal staining of viral antigen and staining of viral particles budding from the neuronal membrane in wild-type vesicular stomatitis virus infection, whereas diffuse membrane and cytoplasmic staining with no budding virus was observed in ts G31 infection. Such findings suggest rapid viral assembly and release of viral particles from cells infected with wild-type virus. In contrast, maturation of ts G31 appears defective, and this would lead to accumulation of viral antigen in the cytoplasm of infected cells. These results correlate with studies in neuroblastoma cells which investigated the growth cycles of wild type, ts G31, and the spinal cord isolate of ts G31 as well as the viral protein-synthetic capacity of these viruses.     

Influenza virus morphogenesis and budding

Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major sub-viral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins.Budding processes involve bud initiation, bud growth and bud release. The presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching-off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.     

Microscopy of internal structures of Sendai virus associated with the cytoplasmic surface of host membranes

The cytoplasmic surface (PS) of the plasma membrane of cells infected with Sendai virus was studied by immunofluorescence microscopy and freeze-drying electron microscopy. After cells had been attached to glass coverslips, they were subjected to a jet stream of physiological buffer which sheared off the upper portion of each cell, leaving the attached membrane with the PS exposed. This uncapping maneuver permitted direct examination of internal virus-specific elements associated with the inner surface of the host cell. At a stage of infection at which viral budding occurs, strands of nucleoprotein (RNP) were observed to be attached to the PS of plasma membranes. The sites at which RNP was adherent to the membrane were modified by virus-specific particles arranged in orthogonal patterns. The presence of the same crystalline structures in the hydrophobic domain of freeze-fractured membranes indicated that they were inserted into the inner lipid leaflet. The spatial association of the surface glycoprotein spikes and the internal RNP with this crystalline structure suggests its special relationship if not identity with the internal viral matrix (M) protein. The possible significance of the localization and crystalline nature of this structural element with respect to viral morphogenesis, hemolytic and cell-fusing activities is discussed. In contrast to the foregoing changes observed in the infected cell, no detectable viral antigens were found on the PS of normal cells to which exogenous virions had been fused. Absence of internalized antigen from the PS under these circumstances could indicate that infectious viral components are processed by the potential host cell in a manner which differs from what is observed with human erythrocytes. In the latter instance internalized antigens after fusion of virus to the cell remain associated with the PS.     

Influenza virus assembly and budding

Influenza A virus causes seasonal epidemics, sporadic pandemics and is a significant global health burden. Influenza virus is an enveloped virus that contains a segmented negative strand RNA genome. Assembly and budding of progeny influenza virions is a complex, multi-step process that occurs in lipid raft domains on the apical membrane of infected cells. The viral proteins hemagglutinin (HA) and neuraminidase (NA) are targeted to lipid rafts, causing the coalescence and enlargement of the raft domains. This clustering of HA and NA may cause a deformation of the membrane and the initiation of the virus budding event. M1 is then thought to bind to the cytoplasmic tails of HA and NA where it can then polymerize and form the interior structure of the emerging virion. M1, bound to the cytoplasmic tails of HA and NA, additionally serves as a docking site for the recruitment of the viral RNPs and may mediate the recruitment of M2 to the site of virus budding. M2 initially stabilizes the site of budding, possibly enabling the polymerization of the matrix protein and the formation of filamentous virions. Subsequently, M2 is able to alter membrane curvature at the neck of the budding virus, causing membrane scission and the release of the progeny virion. This review investigates the latest research on influenza virus budding in an attempt to provide a step-by-step analysis of the assembly and budding processes for influenza viruses.     

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with α-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170000 and 24000 daltons) and a polypeptide of 52000 daltons. These results suggesl lhal the ionophore inhibits assembly of a virus which malures at intracellular membranes.     

Membrane changes associated with assembly of visna virus.

The maturation of visna virus at the surface membranes of sheep choroid plexus cells has been studied by freeze-fracture, deep-etching, surface replication, and scanning EM techniques. The results were integrated with data obtained from thin sections. The first changes detectable in the membranes of infected cells consist of flat regions which lack intramembrane particles and show clusters of globular units on corresponding areas of the surface. Protrusion of the cell membrane, characteristic of the budding process, appears to be initiated after a capsid attaches to these modified membrane regions. During the growth of the viral bud, the areas lacking intramembrane particles, the crescent capsids, and the surface covered with globular units increase in size simultaneously. The surface units become more widely spaced as if they were anchored to the bending capsid. At the end of this development, viruses bud off singly or in rows from the soma and long processes of the infected cells. The capsid detaches from the envelope and forms a central core, the surface units become larger and more closely apposed, and free viruses become smaller than viral buds. Thus, visna virus formation requires insertion and growth of surface units probably representing viral-coded proteins, reorganization of host cell membrane proteins under these units, and, initially, attachment of a coiled capsid to the modified membrane.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

The exit of vaccinia virus from infected cells

Vaccinia virus (VACV) is the prototypic member of the Poxviridae a group of large DNA viruses that replicate in the cell cytoplasm. The entry and exit of VACV are complicated by the existence of two distinct forms of virus, intracellular mature virus (IMV) and extracellular enveloped virus (EEV), that are surrounded by different numbers of lipid membranes and have different surface proteins. Here the mechanisms used by these different forms of VACV to leave the infected cell are reviewed. Whereas some enveloped viruses complete virus assembly by budding through the plasma membrane, infectious poxvirus particles (IMV) are produced within the cytoplasm. These particles are either further enveloped by intracellular membranes to form intracellular enveloped virus (IEV) that are transported to the cell surface on microtubules and exposed on the cell surface by exocytosis, or are released after cell lysis. If the enveloped virion remains attached to the cell surface it is called cell-associated enveloped virus (CEV) and is propelled into surrounding cells by growing actin tails beneath the plasma membrane. Alternatively, the surface virion may be released as EEV.     

Role of multivesicular bodies and their components in the egress of enveloped RNA viruses

As an enveloped virus buds, the nascent viral capsid becomes wrapped in a plasma membrane-derived lipid envelope, and a membrane fission event is thus necessary to separate the virion from the host cell. This membrane fission event is well characterised in the case of enveloped RNA viruses, where it is promoted by late assembly domains (L-domains) present at the level of specific viral structural proteins. Research conducted over the past 10 years has demonstrated that L-domains represent docking sites for cellular proteins essential for the biogenesis of a cellular organelle, the multivesicular body (MVB). In this way, enveloped RNA viruses hijack the MVB components to the cellular site where the budding is executed. This review will focus on the cellular machinery exploited by enveloped RNA viruses in order to be released from infected cells. The role of ubiquitin and lipids in viral budding will also be discussed.     

A novel method for analysis of membrane microdomains: vesicular stomatitis virus glycoprotein microdomains change in size during infection,and those outside of budding sites resemble sites of virus budding

Membrane proteins, including viral envelope glycoproteins, may be organized into areas of locally high concentration, commonly referred to as membrane microdomains. Some viruses bud from detergent-resistant microdomains referred to as lipid rafts. However, vesicular stomatitis virus (VSV) serves as a prototype for viruses that bud from areas of plasma membrane that are not detergent resistant. We developed a new analytical method for immunoelectron microscopy data to determine whether the VSV envelope glycoprotein (G protein) is organized into plasma membrane microdomains. This method was used to quantify the distribution of the G protein in microdomains in areas of plasma membrane that did not contain budding sites. These microdomains were compared to budding virus envelopes to address the question of whether G protein-containing microdomains were formed only at the sites of budding. At early times postinfection, most of the G protein was organized into membrane microdomains outside of virus budding sites that were approximately 100-150 nm, with smaller amounts distributed into larger microdomains. In contrast to early times postinfection, the increased level of G protein in the host plasma membrane at later times postinfection led to distribution of G protein among membrane microdomains of a wider variety of sizes, rather than a higher G protein concentration in the 100- to 150-nm microdomains. VSV budding occurred in G protein-containing microdomains with a range of sizes, some of which were smaller than the virus envelope. These microdomains extended in size to a maximum of 300-400 nm from the tip of the budding virion. The data support a model for virus assembly in which G protein organizes into membrane microdomains that resemble virus envelopes prior to formation of budding sites, and these microdomains serve as the sites of assembly of internal virion components.     

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.     

Replication of Mayaro virus in Aedes albopictus cells: an electron microscopic study

Summary The replication of Mayaro virus inAedes albopictus cells, was studied by electron microscopy at various times post-infection. In infected cells we observed the presence of cytoplasmic vesicles containing viral nucleocapsids and mature virus particles but at no time did we detect virus budding into such vacuoles. Budding of virus through plasma membrane was rarely observed. Our results are discussed considering the possibility of the release of virus particles to the extracellular space by exocytosis.     

Assembly of enveloped respiratory syncytial virus particles within the cytoplasm of infected vero cells

Summary Electron microscopic examination of syncytia induced by a bovine respiratory syncytial virus strain in Vero cell cultures revealed the presence in the cytoplasm of assembled enveloped virus particles within inclusions of variable sizes. Moreover, budding virus particles were shown occasionally in the intracytoplasmic vesicles. These particles were filamentous in form, about 80–120 nm in diameter, variable in length, containing 12 nm diameter nucleocapsids, and looked like the extracellular particles budding at the plasma membranes. This is the first report on assembly of intracellular virus particles in cells infected by a member of the familyParamyxoviridae. Vero cell-dependent variations appear to be the factor leading to the defect in the late virus replication cycle.     

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm.Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.     

Morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells.

The morphogenesis of porcine rotavirus was similar in vitro in porcine kidney (PK) cell cultures and in vivo in porcine epithelial cells as examined by electron microscopy. Infected cells contained cytoplasmic, non-membrane-bound viroplasm and accumulations of virus particles within cisternae of the rough endoplasmic reticulum (RER). Three types of virus particles were noted: double-shelled or complete particles which averaged 77 nm in diam.; single-shelled or naked particles which ranged from 50 to 55 nm in diam.; and electron-dense nucleoids, or cores, 31 to 38 nm in diam. Virus particles acquired outer shells by budding through either matrices of granular, electron-dense viroplasm or membranes of distended RER. Accumulation of numerous single-shelled particles was observed only in PK cell cultures containing a high percentage of infected cells. In these cells, virus release occurred through disruption of the plasma membrane. Tubules, similar in diameter to the single-shelled particles, were observed in the nuclei of a few infected PK cells.