Paramyxoviruses were used for many of the early membrane fusion experiments. The paramyxoviruses being discussed have two glycoproteins that appear as spikes on the virus. One glycoprotein is the HN protein, which binds receptors that contain N
-acetylneuraminic acid and which has neuraminidase activity. The second glycoprotein is the F (f
usion) protein, which is required for membrane fusion to occur. The M protein, which is thought to play a major role in virus assembly, is apposed to the inner side of the viral membrane. The M protein is small (Mr
, ~38,500 to 41,500), basic, and somewhat hydrophobic but does not have a hydrophobic region long enough to go across a lipid bilayer. It is probably important in membrane uncoating. Paramyxoviruses have single-stranded RNA of negative polarity, which associates with the nucleocapsid (N) protein to form a helical ribonucleoprotein (RNP). The L and P proteins, which together form RNA-dependent RNA transcriptase, are associated with the RNP (34
Until the end of the 1970s, studies of viral membrane fusion and uncoating often used paramyxoviruses that were grown for several days on the chorioallantoic membranes of chicken eggs with 9- to 11-day-old embryos. The chorioallantoic membrane is a highly vascular, fused layer of chorion and allantois and can be dissected from the egg. The chorioallantoic membrane cells produce infectious viruses with the F0 protein cleaved into F1 and F2 proteins. This cleavage is necessary before the F protein is active and the virus can fuse its membrane.
Many of the paramyxoviruses cause hemagglutination by binding red cells. Hemagglutination may be followed by hemolysis, which can be easily measured by recording the optical density at 575 nanometers (OD575) to measure hemoglobin release. As discussed below, hemolysis requires membrane fusion, but membrane fusion does not necessarily result in hemolysis.
Often, Sendai virus (SeV) was the paramyxovirus used. It is a mouse parainfluenza virus type 1 and is also is referred to as hemagglutinating virus of Japan (HVJ). It takes between 14 and 20 h at 36°C for one round of replication on chorioallantoic membrane cells in embryonated chicken eggs. Sendai viruses grown for 24 h are called “early-harvest” viruses. If the viruses are not harvested after 1 day in the eggs, the progeny viruses infect additional chorioallantoic cells, and the number of viruses produced increases considerably. Therefore, viruses grown in eggs for 2 to 3 days were commonly used in early times. The viruses grown 48 h or longer are termed “late-harvest” viruses (20
). Early-harvest virus preparations cause a low level of hemolysis, and some do not cause hemolysis at all. As the time of viral growth in eggs increases, the amount of hemolysis produced by the virus increases. In a late-harvest virus preparation, there are always viruses that were released just before harvest, and so there are always some viruses that have the attributes of early-harvest virus. In early-harvest preparations, there are usually some viruses that are damaged and behave like late-harvest viruses. Thus, a virus preparation is usually not purely one or the other kind of virus, but it is possible to make preparations that contain predominantly early-harvest (intact) or late-harvest (damaged) viruses.
There are considerable morphological differences between early-harvest and late-harvest Sendai viruses (HVJ) (26
). These differences are shown in Fig. . Negatively stained early-harvest viruses (Fig. ) have a relatively homogeneous size and are oval shaped. Thin sections of early-harvest virus (Fig. ) show that the RNP is regularly folded parallel to the long axis of the virions. If the virus is sectioned at right angles to the long axis (arrow), the strands of RNP are seen in cross-section and are distributed in an orderly fashion at about equal distances from each other and mainly parallel to the virus axis. A few damaged viruses are also seen in this section. The late-harvest viruses (Fig. ) are pleomorphic, with many spherical viruses of various sizes. Many late-harvest viruses have RNP strands that are randomly distributed in the virus rather than regularly arranged in relation to the membrane. The viruses within the late-harvest preparation that have an early-harvest or intermediate virus morphology are indicated by the arrows. The internal volumes of the larger late-harvest virus are multiples of the internal volume of early-harvest virus. Therefore, it is not surprising that late-harvest viruses have lost considerable internal organization.
FIG. 1. Intact (“early-harvest”) and damaged (“late-harvest”) HVJ (Sendai virus). (A) Thin section of late-harvest HVJ (Sendai virus). Arrows indicate the early-harvest viruses present among the viruses in a late-harvest preparation. (more ...)
Freeze-fracture electron microscopy has been used for studying membrane structures (4
). The sample is rapidly frozen, fractured, and then shadowed. Intrinsic membrane proteins are seen in fractured membranes as intramembranous particles (IMPs) in the smooth lipid part of the membrane. The exoplasmic face (E face or EF) represents the hydrophobic side of the outer monolayer. The protoplasmic face (P face or PF) represents the hydrophobic side of the inner monolayer. Late-harvest viruses have IMPs with a mode diameter of 150 Å in the E face, whereas early-harvest viruses rarely show IMPs (26
If early-harvest viruses are purified and concentrated and then incubated for 2 days at 37°C in allantoic fluid, they develop IMPs and an appearance consistent with that of late-harvest viruses. The same is true if purified early-harvest viruses are left for 2 days at 4°C with balanced salt solution, pH 7.6. LLC-MK2 cells begin to release F0
HVJ (Sendai virus) at 10 h, and the viral number increases until 3 days of incubation. HVJ grown for 3 days in LLC-MK2 cells have the electron micrograph appearance of late-harvest virus, and freeze-fracture electron microscopy shows IMPs in the E face. Thus, the viruses seem to “age” either during a long period with cells or during a long time in different media without cells. At present, it is not known which membrane protein(s) is altered during aging to result in late-harvest virus. The authors of a study of early-harvest and late-harvest viruses suggested that the M protein could be involved (26
). In the virion, the M protein is associated with the nucleocapsid and with the glycoprotein spikes and is important in viral assembly (34
), and so involvement of the M protein in changes causing late-harvest virus properties seems a good possibility. Noncovalent intermolecular interactions in Sendai virus and Newcastle disease virus were studied with reversible cross-linkers that had a 1.1-nm separation between their functional groups. Nucleoprotein and M protein heterodimers resulting from cross-linking were most prevalent in early-harvest viruses (37
). Not all viruses are as fragile as Sendai virus. However, since Sendai viruses were used for many of the early studies of viral entry, their fragility is an important reason for ideas about membrane uncoating.
If early-harvest viruses are treated with sonication or with repeated freeze-thaw cycles, the amount of hemolysis they produce increases (20
). Measurements of infectivity and hemolysis show that the infectivity goes down after each freeze-thaw cycle as the amount of hemolysis a virus causes increases. Late-harvest viruses produce considerable hemolysis. The sensitivity to chemical or physical manipulations, such as freezing and thawing, seems to exist to some degree for many kinds of viruses. This sensitivity to laboratory proceedings is sometimes also reflected in viral properties after different purification procedures. Experiments with vesicular stomatitis virus and preliminary experiments with simian virus 5 (SV5; parainfluenza virus 5) and influenza virus indicated that the viruses appropriately swelled or shrank with the change in the osmolarity of the external media (3
). Below are discussed the data that show that damaged Sendai virus uncoats in a process that is different from that used by intact virus.
Electron microscopic studies of late-harvest Sendai virus membrane fusion and membrane uncoating with chorioallantoic cells and with L cells.
The 1968 study by Morgan and Howe (41
) included excellent electron micrographs that showed fusion of Sendai virus membranes with the membranes of chorioallantoic cells and of L cells. The continuity of the viral and host membranes was unequivocally clear, so that fusion between Sendai virus membrane and host cell plasma membrane was generally accepted as the mechanism of Sendai virus entry. Their virus was grown in chorioallantoic cavities of 11-day-old chicken embryos for an unstated amount of time. The authors comment on “pleomorphism” of the viruses, and the viruses have the appearance of late-harvest viruses.
Figure A shows a virus that has been incubated with cells at 37°C for 6 min and has fused its membrane with the host plasma membrane. When Sendai virus binds many cell receptors, the cell membrane becomes indented to surround the bound part of the virus. At the edge of the region of binding, the host membrane returns to its normal curvature and in doing so produces a rim around the indentation where the membrane is very curved. Many of the micrographs in the Morgan and Howe study (41
) show viruses that had fused their membranes with host membranes in the vicinity of the very curved membrane edge at the border of a region of binding.
FIG. 2. Membrane fusion and uncoating of damaged and intact Sendai virus. (A) Damaged Sendai virus that has fused with the membrane of a chorioallantoic cell after 1 h at 4°C and 6 min at 37°C. (B) Damaged Sendai virus that has fused with the (more ...)
Figure shows a fused virus and cell with the virus structure widening as the viral membrane merges with the host membrane. Much of the viral contents have mixed with the cytoplasm below the region of fusion. In some micrographs, the contents of the virus and of cell are almost completely mixed. This supported the idea that after fusion of viral and host membranes, there is extensive mixing of the viral and host components.
Even in late-harvest preparations, there are a few early-harvest viruses. Morgan and Howe presented a micrograph wherein the viruses are small and oval and appear to be early-harvest viruses (Fig. ). The ribonucleoprotein (RNP) is very organized and aligned with the long axis of the virus. The virus on the right is bound but not fused with the host and appears to be cut at right angles to the virus axis. The virus on the left in this micrograph has fused with the cell membrane. A connecting structure is present, and the RNP has moved a short distance down the connecting structure into the cell, yet the rest of the virion is intact. This was not commented upon, but at the time, the idea of early-harvest and late-harvest viruses had not yet been proposed. This micrograph raises the question of whether for intact viruses the RNP moves through a connecting structure (connector) without dismantling the viral coat.
Sendai viruses fuse with red cells and may cause hemolysis.
Colloid osmotic swelling of red cells produces hemolysis. Red cells have hemoglobin and do not have the membrane structures, such as microvilli, which allow most cells to expand considerably. Late-harvest Sendai viruses produce a change in the plasma membrane permeability that leads to the osmotic swelling. The increase in permeability is thought to be due to the presence of the virus glycoproteins (probably the F protein) in the red cell membrane. Zn2+
inhibit the changes in permeability. The damage to membranes by hemolytic viruses has been compared to the damage performed by toxins, such as Staphylococcus aureus
alpha-toxin, melittin, complement, and other cytotoxic agents (2
). Virus-induced hemolysis requires both membrane fusion and subsequent changes in membrane permeability.
Changes in membrane permeability also occur in host cells at the beginning of infection with late-harvest viruses, but the host cells do not lyse. The potential across the membrane of FL cells (a line from human amnion) was determined before and after infection with HVJ (Sendai virus) (42
). After incubation with what appears to be late-harvest virus at 37°C, the FL cell membrane potential dropped. The membrane potential was repaired after membrane fusion was completed. The increase of membrane permeability of mouse-grown Lettrée cells seen with late-harvest Sendai virus does not occur after infection with early-harvest virus (54
Knutton studied fusion of nonhemolytic Sendai viruses (grown 24 h or early harvest) with erythrocytes (30
). Scanning microscopy showed that by 2 to 3 min at 37°C, nonhemolytic viruses had fused with the red cell. Many of the nonhemolytic fused viruses do not become completely incorporated into the red cells even after 20 to 30 min of incubation. Hemolytic viruses (grown 72 h or late harvest), however, merge into red cells and cause a change in red cell permeability, which leads to cell swelling and visible red cell-red cell fusion.
Knutton and Bächi used nonhemolytic viruses and hemolytic viruses to investigate the movement of Sendai viral antigens in red cell membranes after membrane fusion (31
). All their batches of “nonhemolytic” virus showed some hemolysis. Unless stated otherwise, the virus they used had a hemolytic figure of 5% or less. They used monolayers of red cells. They detected the Sendai glycoproteins with fluorescein isothiocyanate (FITC) anti-Sendai virus antibody (or with FITC anti-Sendai virus antibody Fab fragments, which gave similar results). When hemolytic or nonhemolytic viruses were added to red cells at 4°C, the viruses did not fuse because of the temperature, and the immunofluorescence of viral antigens was granular. After 10 min at 37°C, the immunofluorescence of the hemolytic viruses became diffused in red cells, and after 1 to 2 h, all red cells showed uniform fluorescence. When nonhemolytic viruses were added to red cells, the immunofluorescence maintained the grainy pattern initially present at 4°C even after 1 h at 37°C. Therefore, the fused nonhemolytic viruses did not release their membrane glycoproteins from the fused virion membranes into the cell membranes.
Sendai viruses enter ganglioside-containing liposomes.
Sendai viruses, strain ESW5, were incubated in the chorioallantoic sac of eggs for about 68 h, so the virus used was late harvest. The liposome composition was roughly similar to the lipids in BHK-21-F cell plasma membranes (28
), and gangliosides, which contain neuraminic acid, were included to act as receptors (12
). At 37°C, Sendai virus can make a ganglioside-containing liposome envelop it as in phagocytosis (although without vacuole closure) (16
) and can fuse with liposomes (14
). After incubation of viruses and liposomes for 2 h at 37°C, the viral RNP was deposited inside the outer layer of a multilamellar vesicle (MLV), and the viral membrane glycoproteins spread over the surface of the liposome in patches of spikes. This was consistent with results of the Morgan and Howe study of Sendai virus entry into cells. Viruses that have been incubated at 37°C with liposomes for only 1 min show fusion only at the very curved leading edge of the region where the liposome binds the virus (17
). This would be the rim of the envelopment if the region were viewed in three dimensions (3D). Curvature facilitates the close approach of surfaces (13
). In a curved bilayer, the heads of the phospholipids of the outer layer of the bilayer are pulled away from each other, facilitating membrane fusion. Further, the membrane tension of the unbound membrane is on one side of the curvature and virus binding is on the other side, so the membrane between the two regions is probably mechanically stressed. With continued time of incubation after membrane fusion, the remaining binding between the fused virus and liposome decreases until the only remaining contact between virus and liposome is the fusion region. Since viral binding alters the properties of a region of the host membrane, it is possible that a major role of the viral F protein is to make changes in the properties of a region of the viral membrane.
Not all viruses merged into liposomes after fusion of the viral and liposomal membranes. Some fused viruses were in the process of what Morgan and Howe (41
) described as uncoating. However, there were some cases in which the viruses had fused but the viral membrane glycoproteins had not diffused into the liposomes and the virion had not merged into the liposomes (17
). This indicates that membrane uncoating does not necessarily occur immediately after membrane fusion and suggests the possibility of two populations of virus. Preliminary experiments with early-harvest viruses suggested that after membrane fusion, these viruses do not merge into liposomes (15
), but further experiments are needed.