Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Sendai virus M protein binds independently to either the F or the HN glycoprotein in vivo.

We have analyzed the mechanism by which M protein interacts with components of the viral envelope during Sendai virus assembly. Using recombinant vaccinia viruses to selectively express combinations of Sendai virus F, HN, and M proteins, we have successfully reconstituted M protein-glycoprotein interaction in vivo and determined the molecular interactions which are necessary and sufficient to promote M protein-membrane binding. Our results showed that M protein accumulates on cellular membranes via a direct interaction with both F and HN proteins. Specifically, our data demonstrated that a small fraction (8 to 16%) of M protein becomes membrane associated in the absence of Sendai virus glycoproteins, while > 75% becomes membrane bound in the presence of both F and HN proteins. Selective expression of M protein together with either F or HN protein showed that each viral glycoprotein is individually sufficient to promote efficient (56 to 73%) M protein-membrane binding. Finally, we observed that M protein associates with cellular membranes in a time-dependent manner, implying a need for either maturation or transport before binding to glycoproteins.     

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells

Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.     

Replication of Sendai Virus: II. Steps in Virus Assembly

Chick embryo fibroblast cultures infected with Sendai virus were incubated with (3)H-uridine in the presence of actinomycin D beginning at 18 hr after infection. The 35 and 18S virus-specific ribonucleic acid (RNA) components were found in a ribonuclease-sensitive form in the cell and appeared to be associated with polyribosomes. Newly synthesized 57S viral RNA was rapidly coated with protein to form intracellular viral nucleocapsid, and no 57S RNA was found "free" (ribonucleasesensitive) in the 2,000 x g supernatant fraction of disrupted cells. The nucleocapsid from detergent-disrupted Sendai virus and that from disrupted cells were indistinguishable in ultrastructure and buoyant density, and neither was found to be infectious or have hemagglutinating activity. Kinetic studies of nucleocapsid and virus formation indicated a relative block in conversion of viral nucleocapsid to complete enveloped virus in these cells, resulting in accumulation of large amounts of nucleocapsid in the cell cytoplasm.     

Purification of measles virus and characterization of subviral components.

Purified measles virus was obtained from [35S]methionine-labeled cells infected at 33 degrees C and maintained in the absence of fetal calf serum. The pellet that was produced by a single high-speed ultracentrifuge spin of culture medium contained virus of purity sufficient for structural analysis. Purified virions contain seven polypeptides with estimated molecular weights of: L, 200,000; G, 80,000; P2, 70,000; NP, 60,000; A, 43,000; F1, 41,000; and M, 37,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Treatment of virions with 0.25% trypsin resulted in a less dense particle which lacked polypeptides G and F1. Solubilization of the viral membrane with the detergent Triton X-100 in low-salt buffer resulted in the loss of the G polypeptide, whereas in the presence of 1 M KCl, Triton X-100 also removed most of the M polypeptide. The nucleocapsids (p = 1.3) obtained from virions treated with Triton X-100 and 1 M KCl contained the L, P2, NP, and M polypeptides. Nucleocapsids isolated from the cytoplasm of infected cells were predominantly composed of the NP polypeptide with smaller amounts of either polypeptide P2 or novel polypeptides, related to NP, with estimated molecular weights of 56,000 to 58,000 and 45,000 to 46,000. A significant amount of polypeptide L was always found in association with nucleocapsids isolated either from virions or from the cytoplasm of infected cells. A membrane component containing the viral membrane polypeptides G, F1, and M was also isolated from infected cells. The data presented here thus suggest that L is an integral part of the nucleocapsid complex. In addition, 37,000-molecular-weight polypeptide (M) appears to have the function described for the matrix proteins of other paramyxoviruses.     

Stereo images of vesicular stomatitis virus assembly.

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.     

Transmembrane communication in cells chronically infected with measles virus

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.     

Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein.

Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.     

Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.     

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.     

Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures.

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.     

Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus.

The association of vesicular stomatitis virus proteins with intracellular and plasma membranes was examined by pulse and pulse-chase labeling of virus-infected HeLa cells with [35S]methionine and separation of cell homogenates into three major membrane fractions in discontinuous sucrose gradients. The glycoprotein G was primarily associated with rough endoplasmic reticulum-like membranes after short radioactive pulses (2 to 4 min) but accumulated in the plasma membrane-enriched fraction and the smooth internal membrane fraction with longer pulse or chase periods. The nucleocapsid protein N and the matrix protein M accumulated in the rough endoplasmic reticulum and plasma membrane-like fractions but not in the smooth internal membrane fraction. Only a fraction (35 to 40%) of the viral protein synthesized during a short pulse in the mid-cycle of infection was apparently utilized in released virus. The newly synthesized virus proteins first appeared in released virus in the order: M, N and L, and G.     

Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins.     

Instability of the viral M protein in BHK-21 cells persistently infected with Sendai virus

The study of viral protein expression in BHK cells persistently infected with Sendai virus showed that the viral M protein was greatly reduced in amount or absent in these cells. Pulse-chase experiments demonstrated that the M protein was synthesized at a normal rate, but was unstable compared to the other viral proteins. The M protein instability was independent of temperature and could account for part of the reduction in viral production by persistently infected cells. When a virus stock was grown in embryonated chicken eggs from viruses produced by persistently infected BHK cells, the M protein of this stock presented a restored stability in BHK cells.     

Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.