Paramyxovirus fusion (F) protein: a conformational change on cleavage activation.

The fusion (F) protein of the paramyxovirus SV5 promotes both virus-cell and cell-cell fusion. Recently, the atomic structure at 1.4 A of an extremely thermostable six-helix bundle core complex consisting of two heptad repeat regions of the F protein has been described (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S Jardetsky, Mol. Cell 3, 309-319, 1999). To analyze the conformations of the F protein at various stages of the membrane fusion process and to understand further the role of formation of the six-helix bundle core complex in promotion of membrane fusion, antibodies to peptides corresponding to regions of the F protein were obtained. Major changes in F protein antibody recognition were found after cleavage of the precursor protein F(0) to the fusogenically active disulfide-linked heterodimer, F(1) + F(2), and antibodies directed against the heptad repeat regions recognized only the uncleaved form. A monoclonal antibody directed against the F protein showed increased recognition at the cell surface of the cleaved form of the F protein as compared to uncleaved F protein, again indicating changes in conformation between the uncleaved and cleaved forms of the F protein. Anti-peptide antibodies specific for the heptad repeat regions were unable to precipitate a synthetic protein that consisted of the heptad repeat regions separated only by a small spacer, suggesting that the antibodies are unable to recognize their target regions when the heptad repeats are present in the six-helix bundle core complex. Taken together, these data indicate that the six-helix bundle core complex is not present in the precursor molecule F(0) and that significant conformational changes occur subsequent to cleavage of the F protein.

Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation.

The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state.

The paramyxovirus SV5 small hydrophobic (SH) protein is not essential for virus growth in tissue culture cells.

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.

The V protein of the paramyxovirus SV5 interacts with damage-specific DNA binding protein.

The simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: V and the phosphoprotein P. The V and P proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of V contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a glutathione-S-transferase (GST)-fusion protein selection of cell lysate assay, the GST-V protein was found to interact with the 127-kDa subunit (DDB1) of the damage-specific DNA binding protein (DDB) [also known as UV-damaged DNA binding protein (UV-DDB), xeroderma pigmentosum group E binding factor (XPE-BF), and the hepatitis B virus X-associated protein 1 (XAP-1)]. A reciprocal GST-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and V interact, and it was found that V and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing V and DDB1 using the vaccinia virus T7 expression system. The interaction of V and DDB1 involves the carboxyl-terminal domain of V in that either deletion of the V carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The V proteins of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in GST-fusion protein selection assays using in vitro transcribed and translated DDB1.

The RNA binding region of the paramyxovirus SV5 V and P proteins.

The V/P gene of simian virus 5 (SV5) encodes two proteins, V (222 residues) and the phosphoprotein P (392 residues). The V and P proteins are amino coterminal for 164 residues, but they have unique carboxy termini due to addition of two nontemplated G residues to the P mRNA during transcription. We have shown that the V and P proteins bind RNA by using both Northwestern blot analysis and ultraviolet-light crosslinking. The RNA-binding region has been mapped to a region in the P and V proteins which contains five basic residues (K74, K76, K77, R79, K81). Either deletion of the basic residues or substitution of the basic residues with alanine inhibited RNA binding by the V or P proteins.

Paramyxovirus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein interactions: intracellular retention of F and HN does not affect transport of the homotypic HN or F protein.

To investigate a possible intracellular coassociation of the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins in a living cell, without resorting to chemical crosslinking and antibody coimmunoprecipitation, we tagged the cytoplasmic N-terminus of SV5 HN with a RRRRR motif and HPIV-3 HN with a RRR motif for endoplasmic reticulum (ER) retention. In addition, we tagged the cytoplasmic C-terminus of SV5 and HPIV-3 F with a KK motif. The RRR- or RRRRR-tagged HN molecules were coexpressed in mammalian cells together with the homologous wt F proteins, and the KK-tagged F molecules were coexpressed with the homologous wt HN proteins, and in each case the transport of the wt F or HN molecules was investigated. The data suggest that an association of F and HN of sufficient affinity to alter the transport of the reporter molecule does not occur intracellularly in the ER or the Golgi apparatus.

Recovery of infectious SV5 from cloned DNA and expression of a foreign gene.

A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3' end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals.

Mutants of the paramyxovirus SV5 fusion protein: regulated and extensive syncytium formation.

To study paramyxovirus-mediated cell fusion it would be advantageous to express in a cell a single protein that could cause regulated syncytium formation at neutral pH following a specific activation signal. We have constructed two SV5 fusion (F) protein mutants that contain three arginine residues in the cleavage site and two separate glycine to alanine changes in the fusion peptide. The mutants were expressed in CV-1 cells using an SV40 recombinant virus vector. The mutant F proteins required addition of exogenous trypsin to cleave F0 to F1 and F2. Massive syncytium formation occurred within 2-4 hr following addition of trypsin to the SV40 recombinant F virus-infected CV-1 cells.

The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions.

The paramyxovirus simian virus 5 (SV5) cysteine-rich V protein has been shown to be a virus structural protein by analysis of the polypeptides of purified SV5 virions. In addition, the V protein has been identified as a component of the virus nucleocapsid core both by the analysis of the polypeptides present in radioactively labeled preparations of purified nucleocapsids and by immunoelectron microscopy. Quantitative autoradiography was used to determine that there are approximately 350 molecules of the V protein in virions. The V protein has been purified from V recombinant baculovirus-infected insect cells and by using inductively coupled argon plasma atomic emission spectroscopy it was found that each molecule of V binds two zinc atoms.

Studies with cross-linking reagents on the oligomeric form of the paramyxovirus fusion protein.

The oligomeric form of the paramyxovirus simian virus 5 (SV5) fusion (F) glycoprotein has been examined by using chemical cross-linking and sucrose density gradient fractionation. In addition, chemical cross-linking was used to examine the kinetics of assembly of the F oligomer. Analysis by SDS-PAGE on 3.5% gels of the cross-linked F molecules indicated three major species with calculated molecular weights of M(r) approximately 65, M(r) approximately 130, and M(r) approximately 195 kDa, suggesting F monomers, dimers, and trimers, respectively. The cross-linked F species of M(r) approximately 195 kDa migrated on gels faster than influenza virus hemagglutinin trimers and between the dimeric and tetrameric forms of paramyxovirus hemagglutinin-neuraminidase (HN). Furthermore, the F protein oligomer was found to sediment slower than the HN tetramer on sucrose gradient centrifugation. SDS-PAGE analysis of the cross-linked F protein of Newcastle disease virus and human parainfluenza virus 3 showed a pattern very similar to that found for SV5. The data are consistent with those expected for the paramyxovirus F protein being a homotrimer.

Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia.

The fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the paramyxovirus simian virus 5 (SV5) were expressed individually or coexpressed in CV-1 cells by using SV40-based vectors and recombinant vaccinia viruses. The extent of detectable fusion in a syncytium formation assay was found to be affected by the expression system used. In addition, when HN was coexpressed with F, it was found that the expression vector system influenced the contribution of HN in forming syncytia. The abilities of the SV5, human parainfluenza virus type 3, and Newcastle disease virus F glycoproteins to cause fusion, when expressed alone or coexpressed with HN, were directly compared by using the SV40-based vector system in CV-1 cells. The F proteins exhibited various degrees of fusion activity independent of HN expression, but the formation of syncytia could be enhanced to different extents by the coexpression of the homotypic HN protein.

RNA editing by G-nucleotide insertion in mumps virus P-gene mRNA transcripts.

A guanine nucleotide insertion event has been shown to occur at a specific site within mumps virus P-gene mRNA transcripts. The region of the mRNA containing the site expected to be used for RNA editing and the complementary portion of the genomic RNA were cloned, and their nucleotide sequences were obtained. The genomic RNA was found to possess six C residues at the insertion site, whereas 63% of the P-gene-specific mRNA transcripts were found to have from two to five G residues inserted at this position in the RNA. An unedited mRNA was shown to encode the mumps virus cysteine-rich protein V, and mRNA transcripts containing two and four inserted G residues were translated to yield the mumps virus P and I proteins, respectively.

Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.

Digital teleradiology: Seaforth--London network.

A total digital teleradiology system using a prototype DuPont laser beam scanner (DTR 2000) and a Bell Canada digital transmission network was evaluated. A total of 489 radiographic and ultrasonographic examinations were transmitted for interpretation from a 41-bed rural community hospital in Seaforth to the University Hospital in London, Ontario, 80 km away. There was concurrence in 98% of these examinations, i.e. the laser-sensitive facsimile film clearly duplicated the original film findings and allowed a confident interpretation to be made. We conclude that this system could readily serve the needs of family physicians in rural communities for radiologic consultation, diagnosis, management, and triage of patients 24 hours per day.

Analysis of the relationship between cleavability of a paramyxovirus fusion protein and length of the connecting peptide.

The relationship between the length of the connecting peptide in a paramyxovirus F0 protein and cleavage of F0 into the F1 and F2 subunits has been examined by constructing a series of mutant F proteins via site-directed mutagenesis of a cDNA clone encoding the simian virus 5 F protein. The mutant F proteins had one to five arginine residues deleted from the connecting peptide. The minimum number of arginine residues required for cleavage-activation of the simian virus 5 F0 protein by host cell proteases was found to be four. F proteins with two or three arginine residues in the connecting peptide were not cleaved by host cell proteases but could be cleaved by exogenously added trypsin. The mutant F protein possessing a connecting peptide consisting of one arginine residue was not cleaved by trypsin. The altered F proteins were all transported to the infected-cell plasma membrane as shown by cell surface immunofluorescence or cell surface trypsinization. However, the only mutant F protein found to be biologically active as detected by syncytium formation was the F protein which has four arginine residues at the cleavage site. The results presented here suggest that in the paramyxovirus F protein the number of basic amino acid residues in the connecting peptide is important for cleavage of the precursor protein by host cell proteases but is not the only structural feature involved. In addition, the data indicate that cleavage of F0 into F1 and F2 does not necessarily result in biological activity and that the connecting peptide may affect the local conformation of the F polypeptide.

Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5.

The "P" gene of the paramyxovirus SV5 encodes two known proteins, P (Mr approximately equal to 44,000) and V (Mr approximately equal to 24,000). The complete nucleotide sequence of the "P" gene has been obtained and is found to contain two open reading frames, neither of which is large enough to encode the P protein. We have shown that the P and V proteins are translated from two mRNAs that differ by the presence of two nontemplated G residues in the P mRNA. These two additional nucleotides convert the two open reading frames to one of 392 amino acids. The P and V proteins are amino coterminal and have 164 amino acids in common. The unique C terminus of V consists of a cysteine-rich region that resembles a cysteine-rich metal binding domain. An open reading frame that contains this cysteine-rich region exists in all other paramyxovirus "P" gene sequences examined, which suggests that it may have important biological significance.

Comparison of the relative roles of the F and HN surface glycoproteins of the paramyxovirus simian virus 5 in inducing protective immunity.

To compare the relative roles of the paramyxovirus simian virus 5 (SV5) major surface glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN), in inducing protective immunity, two recombinant vaccinia viruses were constructed. The F and HN polypeptides expressed by the recombinant viruses were indistinguishable from their authentic SV5 counterparts in electrophoretic mobility, glycosylation, and, for the F protein, cleavage of the precursor, F0, to the disulfide-linked subunits F1 and F2. Injection of rabbits and hamsters with live recombinant virus elicited an antibody response to either F or HN and provided a source of monospecific polyclonal antisera to the SV5 proteins. The vaccinia virus-SV5 F (vaccinia-F) recombinant induced higher levels of neutralizing antibody than did the vaccinia-HN recombinant, but animals inoculated with vaccinia-HN were better protected from challenge with SV5. Animals infected with both the vaccinia-HN and vaccinia-F viruses were nearly as well protected from challenge as were animals infected with SV5.

Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor.

The hydrophobic NH2 terminus of F1 (FRED) of the simian virus 5 fusion (F) protein is implicated in mediating cell fusion, but in the inactive F0 precursor the FRED is translocated across membranes. Hybrid proteins containing the FRED as a potential membrane anchorage domain and a mutant of F0 lacking the preceding five-arginine cleavage/activation site were used to study the effect of position on the FRED. The experiments indicate that the SV5 F protein has evolved an exquisite control system for biological activity: the FRED is close to the threshold of hydrophobicity required to function as a membrane anchor. The FRED is not sufficiently hydrophobic to halt translocation when in an internal position, but on cleavage/activation the threshold of hydrophobicity is effectively lowered, and the FRED, now the NH2 terminus of F1, is able to interact stably with membranes.

Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA.

cDNAs encoding the mRNAs for the fusion protein (F) and the hemagglutinin/neuraminidase protein (HN) of the paramyxovirus simian virus 5 have been inserted into a eukaryotic expression vector under the control of the simian virus 40 late promoter. The F and HN proteins synthesized in recombinant infected cells are indistinguishable in terms of electrophoretic mobility and glycosylation from the proteins synthesized in simian virus 5-infected cells. In addition, the expressed F and HN proteins have been shown to be anchored in the plasma membrane in a biologically active form by indirect live cell immunofluorescence, the F-mediated formation of syncytia, and the ability of HN to cause the hemadsorption of erythrocytes to the infected cell surface.

Identification and predicted sequence of a previously unrecognized small hydrophobic protein, SH, of the paramyxovirus simian virus 5.

A previously unrecognized gene (SH) has been identified on the virion RNA of the paramyxovirus simian virus 5 between the genes for the fusion protein and the hemagglutinin-neuraminidase. An SH mRNA of 292 nucleotides (plus polyadenylate residues), transcribed from the SH gene, has been identified. The SH mRNA contains a single open reading frame which encodes a polypeptide of 44 amino acids with a molecular weight of 5,012. The SH polypeptide is predicted to contain an extensive hydrophobic region. This protein has been identified in simian virus 5-infected cells, and it has been shown to be encoded by the SH mRNA by in vitro translation of size-fractionated mRNAs, hybrid-arrest translation, and hybrid-selection translation.