An unusual transmissible agent--MaTu.

MaTu is an agent, believed to be derived from a human mammary carcinoma, which displayed several extraordinary properties. These were: RIP and PAGE revealed in MaTu-infected cells only a single protein band of Mr 58 k, the gp 58. This gp 58 was immunoprecipitated by antibodies present in some human sera as well as in some sera of rabbits, sheep, and cattle. MaTu had an extremely restricted host range: it was transmissible only to HeLa cells, but not to human embryo fibroblasts, to three human tumour cell lines (T 47 D, T 24, and HMB 2) or to monkey Vero and rabbit SIRC cells. A retrovirus with a broad host range, used as a helper (X-MLV) enabled the transmission of MaTu to human fibroblasts, but not to Vero or SIRC, which are also permissive for X-MLV. These observations, together with our previous reports, support the view that MaTu might either be a novel type of defective virus, or even a non-viral autonomous genetic element.

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein.

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.

Vesicular stomatitis virus M protein in the nuclei of infected cells.

The M protein of vesicular stomatitis virus (VSV) was localized in the nuclei and cytoplasm of VSV-infected cells by subcellular fractionation and immunofluorescence microscopy. Nuclei isolated from VSV-infected Friend erythroleukemia cells were fractionated into a nuclear membrane and a nucleoplasm fraction by DNase digestion and differential centrifugation. G protein was present in the membrane fraction, and M protein was present in the nucleoplasm fraction. Immunofluorescence detection of M protein in the nucleus required that fixed cells be permeabilized with higher concentrations of detergent than were required for detection of M protein in the cytoplasm of VSV-infected BHK cells.

NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin.

The phosphoprotein (NS) of vesicular stomatitis virus is an indispensable subunit of the virion-associated RNA polymerase (L). NS consists of a highly acidic NH2-terminal domain and a basic COOH-terminal domain. Unlike the latter, the amino acid sequences of the NH2-terminal regions are highly dissimilar among different viral serotypes, although they share structural similarities. We have cloned an NS gene into the SP6 transcription vector and replaced the 5'-terminal 80% by a full-length gene for beta-tubulin, which contains an acidic COOH-terminal domain. Here we present evidence that the chimeric tubulin-NS protein is biologically active and that the acidic region in tubulin directly affects the transcription reaction. These observations indicate that NS probably functions as an activator protein in which the acidic domain stimulates transcription of the viral genes by interacting with the RNA polymerase as observed for eukaryotic cellular transcription activators.

The functional domains of the phosphoprotein (NS) of vesicular stomatitis virus (Indiana serotype).

A full-length cDNA clone of the mRNA encoding the phosphoprotein (NS) of the Indiana serotype of vesicular stomatitis virus was inserted into the SP6 transcription vector. By in vitro transcription of the inserted gene followed by translation of the mRNA in a rabbit reticulocyte lysate, NS protein was synthesized. The biological activity of the protein was demonstrated by RNA synthesis in vitro by reconstitution with L protein and N-RNA template purified from virions. Using oligonucleotide-directed RNase H cleavage of the full-length NS mRNA, a series of deleted RNAs were made which gave rise to corresponding size classes of truncated NS protein after translation in vitro. The N-RNA template binding site was located at the C-terminal domain (21 amino acids) of the NS protein and the L-protein binding site was present within 14 amino acids spanning the NH2-terminal side of the N-RNA binding site. These results are similar to that obtained with the NS protein of the New Jersey serotype of VSV, indicating conservation of the functional domains within the VSV serotypes.

Polarity of the Forssman glycolipid in MDCK epithelial cells.

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.

Sequences of Chandipura virus N and NS genes: evidence for high mutability of the NS gene within vesiculoviruses.

The nucleotide sequence of the 3' end of the genome of Chandipura (CHP) virus, including the complete sequences of the nucleocapsid (N) and phosphoprotein (NS) genes was determined, principally from cloned cDNAs of the N and NS mRNAs. The NS mRNA of CHP virus is 908 bases in length and encodes a protein of 293 amino acids. Comparison of the CHP virus NS protein sequence with those of vesicular stomatitis virus of the New Jersey serotype (VSV (NJ)) and of the Indiana serotype (VSV (IND] revealed homologies of only 23 and 21%, respectively, with no consecutive stretches of more than four amino acids identical among the three sequences. As with the two VSV serotypes, the highest homology between the NS proteins of CHP and VSV was in a 20-amino acid region near the carboxy termini of the proteins. Of the potential phosphorylation sites, there are eight conserved serine or threonine residues among the three sequences. Despite the dissimilarity among primary sequences of the NS proteins, their overall structure, as assessed by amino acid composition and by the relative hydropathicities of the sequences, has been conserved throughout evolution. The N mRNA of CHP virus is 1291 bases long and encodes a protein of 422 amino acids. In contrast to the NS protein, the CHP N protein is at least 50% homologous to the N proteins of each of the VSV serotypes. We have identified a region near the center of these N protein sequences which is conserved among members of both the rhabdovirus and paramyxovirus families. This extent of conservation of the N protein sequences underscores the high rate of mutability of the NS protein sequences among the vesiculoviruses.

Conservation of potential phosphorylation sites in the NS proteins of the New Jersey and Indiana serotypes of vesicular stomatitis virus.

A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31 000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15% difference at the nucleotide level and 10% difference at the amino acid level; the majority of the changes were located in the 3' half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50% nucleotide and 33% amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity.

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies.

The matrix (M) protein of vesicular stomatitis virus (VSV) appears to function as a bridge between the ribonucleocapsid (RNP) core and the envelope in assembly of the virion. Two such properties would necessitate at least one site for interaction with the nucleocapsid and one with the envelope. In this study M protein was found to mediate the in vitro binding to RNP cores of phospholipid vesicles, representing membrane structures. The M protein could bind initially to either the vesicles or the RNP cores to promote RNP-vesicle association. A trypsin-resistant fragment (MT) of M protein, missing the initial 43 amino acids from its amino terminus, reconstituted with acidic phospholipid vesicles with the same binding efficiency as did whole M protein, suggesting that the carboxy-terminal 81% retained those regions of the M protein which interact with a lipid bilayer. The MT protein, however, was considerably less efficient than intact M protein as an inhibitor of in vitro virus transcription; almost 2.5-fold more MT protein than intact M protein was required for 50% inhibition of VSV transcription, indicating that a site for interaction with the RNP core may have been lost. A monoclonal antibody which is able to reverse the in vitro inhibition of transcription by M protein did not react by immunoblotting with MT protein. Partial tryptic digests of the M protein probed with this monoclonal antibody indicated that epitope 1 lies between amino acid residues 18 and 43. This region appears to be a site that promotes interaction of the M protein with the RNP core of VSV. Monoclonal antibodies to epitopes 2 and 3, which exhibit some overlap in binding to M protein but do not reverse transcription inhibition, were mapped by cleavage with N-chlorosuccinimide at regions in a carboxy direction from epitope 1.

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.

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

Monoclonal antibodies to the M protein of vesicular stomatitis virus (Indiana serotype) and to a cDNA M gene expression product.

Twenty-nine independent hybridomas producing monoclonal antibodies to the matrix (M) protein of vesicular stomatitis virus (Indiana serotype) were prepared by fusion of SP2/0 myeloma cells with spleen lymphocytes obtained from BALB/c mice which had been immunized with the purified M protein. The specific reactivity of each monoclonal antibody was determined by an enzyme-linked immunosorbent assay and a competitive binding assay. Most of the antibodies were of the immunoglobulin G2a and G2b isotypes, although some were immunoglobulin M. By measuring the competitive binding of 125I-antibody, we identified four antigenic determinants in the M protein of the virus; two of these determinants, however, exhibited a large degree of overlap. Western blot analysis revealed little or no cross-reactivity of the antibodies with other viral proteins or with the M protein of the New Jersey serotype. Prolonged trypsin proteolysis removed the first 43 amino acids from the amino-terminal region of the M protein, but it retained its reactivity with monoclonal antibodies to each epitope, except for diminished reactivity with one. To aid in future mapping of these epitopes, we inserted a cDNA clone of the mRNA encoding the M protein of vesicular stomatitis virus into an inducible lac expression vector; the M protein produced in the JM103 strain of Escherichia coli under induced conditions was found to be approximately the same size as native M protein and was recognized by the monoclonal antibodies. These monoclonal antibodies and the cDNA clone should be useful for studying the role of M protein in virus maturation and the regulation of viral transcription.

Constitutively phosphorylated residues in the NS protein of vesicular stomatitis virus.

The NS protein of vesicular stomatitis virus is an auxiliary protein in the virus core (nucleocapsid) that plays a role in virus-specific RNA synthesis. NS exhibits a variety of phosphorylated forms, and the degree of phosphorylation correlates with the rate of RNA synthesis. However, chymotryptic peptide mapping has indicated that all forms of NS share a common cluster of phosphorylated residues. To locate these residues in the primary structure of the molecule, we performed a series of residue-specific chemical and enzymatic cleavages and separated radiophosphate-labeled peptides by gel electrophoresis. The data indicate that the constitutively phosphorylated sites in NS molecules reside in the amino-terminal region of the molecule, between residues 35 and 78. The previously reported resistance of the phosphoamino acids in this region to dephosphorylation by exogenous phosphatase suggests that this domain is embedded within the tertiary structure of the molecule or involved in quaternary interactions. In contrast, the amino acid residues that are phosphorylated secondarily, making NS more active in RNA synthesis, reside in more exposed regions of the molecule.

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells.

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

Studies on the development of the charge heterogeneity of the influenza virus glycoproteins.

The heterogeneity in charge of the influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA) is retained, when glycosylation is inhibited by tunicamycin (TM) or 2-deoxyglucose (2-dg). This is in contrast to the charge heterogeneity of the G protein of vesicular stomatitis virus (VSV), which is mainly due to heterogeneous sulfation of the carbohydrate side chains and therefore is abolished by the above mentioned inhibitors of glycosylation. Thus, the charge heterogeneity of influenza virus glycoproteins might be attributable to some as yet unidentified modifications of the polypeptide backbone.

Immunocytochemical localization of vesicular stomatitis virus proteins N and NS with monoclonal antibodies.

The purpose of this paper is to describe the immunocytochemical-localization of N and NS nucleocapsid proteins of vesicular stomatitis virus in the cells throughout the infectious cycle. N protein was detected in the cytoplasm at 2 h after infection and formed small cytoplasmic clusters which progressively increased in size and number. At 5-6 h, it formed large cytoplasmic inclusions. NS protein was detected in the cytoplasm a little later than N protein and showed almost the same immunostaining pattern. However, diffuse background staining of NS protein was identified throughout the cytoplasm by double immunostaining methods. At electron microscopic level, N protein was mostly granular and occasionally organized in strands at 2-3 h. At 5-6 h, numerous immunostained reaction products were organized in strands. The reaction products of NS protein were almost the same as those of N protein with the exception that diffuse background staining was observed. Cos cells, transfected with SV40 vector containing N gene obtained by recombinant DNA technique, showed clusters of N protein, but virtually no strand at electron microscopic levels. The rapid-freezing and deep-etching replica method demonstrated that loosely coiled VSV genome coated with N protein was localized on cytoplasmic sides of cell membranes in the infected cells. These results showed that complete virus genome replication was needed for strand formation of N and NS proteins and suggested that they were bound to VSV genomes in the infected cells.

Caracterizacion antigenica e inmunogenica de varias cepas del serotipo Indiana de estomatitis vesicular aisladas en Brasil / Antigenic and immunogenic characterization of various strains of the Indiana serotype of vesicular stomatitis isolated in Brazil

Antigenic studies performed with vesicular stomatitis (VS) virus strains of the serotype Indiana (IND), Rancharia-Brazil/66 and Riberao-Brazil/78, isolated in the state of Sao Paulo, Brazil in 1966 and 1979, respectively, indicated that they have to be classified within the subtype IND-2 and that they are related to virus strains Salto-Argentina/63 and Cocal-Trinidad/61. The Espinoza-Brazil/77 strain isolated in 1977 in cattle of the state of Minas Gerais is closely related to the Alagoas-Brazil/64 strain, subtype IND-3. Studies of sera from recovered animals and immunodiffusion gel agar test confirmed the results obtained by 50 percent complement fixation tests. Field pathogenicity indicated that strains from the subtype IND-2 only affected horses, while the IND-3 subtype viruses affected cattle to a lower degree in comparison with horses. The northeast states and Minas Gerais, in Brazil, are affected by VS virus subtype IND-3, while in Sao Paulo and Rio Grande do Sul subtype IND-2 strains are identified

Preparation and analysis of the nucleocapsid proteins of vesicular stomatitis virus and sendai virus, and analysis of the sendai virus leader-NP gene region.

A procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3' end of the Sendai virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5' end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo.