Abstract: Closing the Sendai virus C protein open reading frames (rSeV-DeltaC virus) results in the production of virus particles with highly reduced infectivity. Besides, the Sendai virus C proteins interact with Alix/AIP1 and Alix suppression negatively affects Sendai virus like particle (VLP) budding. Similarly, the Sendai virus M protein has been shown to interact with Alix. On this basis, it has been suggested that Sendai virus budding involves recruitment of the multivesicular body formation machinery. We follow, here, the production of SeV particles upon regular virus infection. We find that neither Alix suppression nor dominant negative-VPS4A expression, applied separately or in combination, affects physical or infectious virion production. This contrasts with the observed decrease of SV5 virion production upon dominant negative-VPS4A expression. Finally, we show that suppression of more than 70% of a GFP/C protein in the background of a rSeV-DeltaC virus infection has no effect either on SeV particle production or on virus particle infectivity. Our results contrast with what has been published before. Possible explanations for this discrepancy are discussed.
Abstract: Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.
Abstract: Paramyxovirus genomes contain a linear array of five to ten genes sequentially transcribed by the viral RNA polymerase. mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter located at the genome 3' end. To gain information about the mechanism involved in transcription initiation, a search was carried out for upstream nucleotides required for gs1 and the effects of the gs1 nucleocapsid protein (N) phase context on transcription regulation were determined. For both purposes, tandem promoter mini-genomes carrying a transcription signal ectopically positioned downstream of a replication-only signal were used. The requirement for hygromycin resistance gene expression was used in an attempt to select essential nucleotides within randomized stretches of nucleotides. Nucleotide insertions or deletions were also made on either side of the transcription signal to change its original N phase context in the five remaining possibilities and GFP expression from these modified signals was assessed. Cell cultures resistant to hygromycin treatment were readily obtained following amplification of mini-genomes harbouring randomized sequences. However, selected nucleotides upstream of gs1 could not be identified under conditions where nucleotides within gs1 were selected. In contrast, it was observed that changing the gs1 N phase context progressively decreased transcription by five- to tenfold. These results are discussed in relation to two different mechanisms of transcription initiation.
Abstract: Detergent resistant membranes (DRMs) are the site of assembly for a variety of viruses. Here, we make use of Sendai virus mutant proteins that are not packaged into virus particles to determine the involvement of this assembly for the virus particle production. We found that, in the context of an infection, (1) all the Sendai virus proteins associated in part with DRMs, (2) mutant HN and M proteins not packaged into virus particles were similarly part of this association, (3) after M protein suppression resulting in a significant reduction of virus production, the floatation profile of the other viral proteins was not altered and finally (4) cellular cholesterol depletion did not decrease the virus particle production, although it somehow reduced their virus infectivity. These results led us to conclude that the assembly complex found in DRM fractions does not constitute a direct precursor of virus particle budding.
Abstract: For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the length of an intervening sequence separating the 3' end of Sendai virus minigenomes and a downstream transcription-initiation signal was increased progressively. It was found that transcription, as measured by green fluorescent protein (GFP) expression, decreased progressively in proportion to the increase in length of the intervening sequence. GFP expression correlated well with the levels of GFP mRNA in the cells, as measured by quantitative primer extension and by RNase protection. Thus, mRNA transcription was inversely proportional to the length of the inserted sequence. These data are evidence that the RNA polymerase initiating transcription at the downstream transcription signal somehow sees the distance separating this signal and the template 3' extremity. Implication of this observation for the nature of the Sendai virus RNA polymerase and for the mechanism by which it synthesizes mRNAs or replication products is presented.
Abstract: In the course of a Sendai virus infection, short genome versions (called defective) are frequently produced. These compete with the full length genome for replication (hence called interfering) and decrease the severity of the infection. The portion of the defective genome required for this effect can be limited, much shorter than the size of the defective genome. We postulated then that defective genome size follows from conditions required for their production more than for exerting the protective effect. Therefore, we set up experiments to determine which of the steps involved in their production may require this larger size. We constructed a series of Sendai virus mini-genomes, increasing their size from the smallest possible and we followed their ability to replicate, to amplify through multiples cycles of infection and to be packaged into virus particles, all steps required for their efficient production. We found that the mini-genomes replicated and amplified efficiently regardless of their size. The uptake into virus particles, however, depended on a threshold length. The data suggest no limitation in the ability of the viral RNA polymerase to generate and amplify very short size defective genomes. They however, point to limitations in the ability of short genomes to be packaged into virus particles.
Abstract: The genomic and antigenomic 3' ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3' end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.
Abstract: The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.
Abstract: The Paramyxoviridae form a large family of viruses containing many human and veterinary pathogens for which a need for antiviral treatment is emphasized, particularly following the recent emergence of new viruses. The viral RNA-dependent RNA polymerase constitutes an obvious target for antiviral compounds. An in vitro assay was developed that allows high throughput screening of compounds potentially inhibiting the Sendai virus RNA-dependent RNA polymerase. Screening relies on the detection of the Photinus pyralis luciferase produced in a transcription/translation coupled assay using a mini-replicon virus. It contains an internal control for possible adverse effects of the tested compounds on translation or on luciferase activity. It is estimated that the mini-replicon template produced in one fertilized egg is sufficient to run 5000-10,000 reactions. This assay constitutes a simple, sensitive and easily automated method to perform high throughput screening of Paramyxoviridae RNA-dependent RNA polymerase inhibitors.
Abstract: The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3'-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3'-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis.
Abstract: We report an 18-month-old girl with rapidly progressive subacute sclerosing panencephalitis, whose non immunised mother had measles at the time of delivery. The patient presented with repetitive episodes of myoclonic jerks of the head and arms, followed by a drop of head and trunk with frequent falls. EEG, CSF studies and MRI confirmed the diagnosis. Despite therapy with isoprinosine and valproate, seizure activity continued and she became vegetative within 2 months, with severe spasticity and swallowing difficulties, and died at the age of 28 months. Early age of onset and rapid progression were most likely related to haematogenous in utero acquisition of the measles virus prior to delivery, as well as immaturity of neuronal and immune systems. CONCLUSION: this case emphasises the importance of a high measles vaccine coverage in the population in order to prevent the risk of disease in general and, in particular, gestational measles.
Abstract: The "rule of six" stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3'-OH end (3'-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3'-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.
Abstract: The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glycoproteins in the process of virus assembly and budding are evaluated. Recombinant Sendai virus (rSeV) mutants are generated carrying modifications in the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state (35)[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cell supernatants are measured relative to the extent of the infection, assessed by the intracellular N protein signal. For both the F and HN cytd truncation mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutants, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this threshold is reached up appears to depend on the nature of the F cytd. A minimal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd truncations result in significant reduction of virion production. On the other hand, a normal virion production can take place with an underrepresentation of or, even, an undetectable HN in the particles. The HN uptake in virion is confirmed to depend on the previously proposed cytd SYWST signal (T. Takimoto, T. Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Virol. 72, 9747-9754.).
Abstract: Substitution of Val(113) in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (M(wt )), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA-M(30 ), an M protein carrying Thr(112)Met and Val(113) Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with M(wt). HA-M (30) is shown (i) to bind mainly to membrane fractions, (ii) not to co-precipitate M(wt), as HA-M(wt) does, (iii) to interfere with the binding of nucleocapsids to membranes and (iv) to accumulate in perinuclear regions, in contrast to HA-M(wt ), which is also found at the cell periphery. Such mutants constitute potential tools for the identification of critical steps in paramyxovirus assembly and budding.
Abstract: The role of the negative-stranded virus accessory C proteins is difficult to assess because they appear sometimes as nonessential and thereby of no function. On the other hand, when a function is found, as in the case of Sendai virus, it represents an enigma, in that the C proteins inhibit replication under conditions where the infection follows an exponential course. Furthermore, this inhibitory function is exerted differentially: in contrast to the replication of internal deletion defective interfering (DI) RNAs, that of copy-back DI RNAs appears to escape inhibition, under certain experimental conditions (in vivo assay). In a reexamination of the C effect by the reverse genetics approach, it was found that copy-back RNA replication is inhibited by C in vivo as well, under conditions where the ratio of C to copy-back template is increased. This effect can be reversed by an increase in P but not L protein. The "rule of six" was differentially observed in the presence or absence of C. Finally, a difference in the ability of the replicating complex to tolerate promoter modifications in RNA synthesis initiation was shown to occur in the presence or the absence of C as well. We propose that C acts by increasing the selectivity of the replicating complex for the promoter cis-acting elements governing its activity. The inhibitory effect of C becomes the price to pay for this increased selectivity.
Abstract: Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.
Abstract: A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.
Abstract: From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3' end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3'/5' end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identify cis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1-31 of the antigenome 3' end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.
Abstract: A natural Sendai virus internal deletion defective interfering (DI) RNA, previously shown to encode a truncated NP protein and previously cloned under the control of the T7 RNA polymerase promoter, was expressed from plasmid and shown to replicate in cell tissue culture when the viral proteins NP, P, and L were coexpressed from cloned genes. The efficient replication was dependent on the total length of the RNA to be a multiple of 6 nucleotides, showing that the "rule of six" applied for a DI RNA that has conserved the end sequences of the nondefective viral RNA. Compared to the copy-back H4 DI RNA, the replication efficiency of the internal deletion DI RNA was reproducibly 20-fold lower. Reciprocal exchanges between the minus-strand 3'-end primary sequences of the two DI RNAs showed that the replication efficiency of the derivatives obtained directly correlated with the origin and the extent of the primary sequence. Moreover, some of the derivatives exhibited a replication efficiency comparable to that of the copy-back DI RNA with, however, the ability to transcribe a functional mRNA similar to the internal deletion DI RNA. This indicated that the transcription ability of a viral RNA was not sufficient to explain a low replication efficiency.
Abstract: We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.
Abstract: The matrix (M) protein is viewed as the regulator of paramyxovirus particle assembly and budding. Accordingly it was observed to be mutated, and/or decreased in amount, in cases where virus particle production was significantly reduced. Here, a non-productive [non-defective and defective interfering (DI)] Sendai virus infection of COS cells is presented where virus particle production is abolished in the presence of a normal amount of intracellular M protein. In this infection the haemagglutinin-neuraminidase envelope glycoprotein is shown to be dispensable for virion production, and the fusion (F0) envelope glycoprotein behaves as in a productive infection. The M protein is shown to accumulate in perinuclear patches within the cytoplasm. In contrast, localization in the plasma membrane is observed in productive infections. However in both productive and non-productive infections a significant fraction of M protein is found in association with cellular membranes. The M protein-membrane association is shown to take place in the absence of any other viral component, and the M protein-membrane complex exhibits properties similar to those observed for the integral membrane protein F0. However these properties are distinct from those of the phosphoprotein, which is thought to associate with membranes in a non-specific manner. Concomitant with the cytoplasmic accumulation of M protein and the reduction of virus particle production in this non-productive infection, DI nucleocapsids are shown not to associate with cellular membrane fractions. This is a property which coincides with their poor envelopment in virus particles. Taken together, these data indicate the need for M protein to be recruited at the perinuclear membranes by the nucleocapsids to participate in viral assembly and budding. This view is consistent with a process of viral assembly taking place on internal cytoplasmic membranes rather than at the plasma membrane.
Abstract: In this report we have monitored viral gene expression, both at the RNA and protein level, after the establishment of a long-term persistent infection of Sendai virus. The persistent infection was initially established by infecting BHK cells with a viral stock containing a short (1.4 kb) copy-back DI (DIH4). After over 120 weeks in culture this short copy-back DI had been replaced by two large deletion DIs (approximately 7 and 12 kb) from which was expressed an N-terminally truncated form of the P protein. The mRNA for this protein was detected in cells and the deletion within the P gene was mapped by PCR cloning and sequencing of intracellular nucleocapsid RNA. This truncated P protein (derived by deleting the N-terminal half of the cloned Pwt gene) has already been shown to function as a dominant negative for DI replication when driven by cloned viral genes. Cloning and expression of the truncated P from the long-term persistent infection revealed that this protein had retained the dominant negative phenotype. The presence of such a protein would severely depress viral gene expression and may therefore play an important role in the maintenance of persistence.
Abstract: A small defective Sendai virus RNA was selectively amplified from a virus preparation obtained after serial undiluted passages in embryonated eggs. Preliminary characterization showed that this defective RNA was a true internal deletion defective RNA, containing the 5' and 3' ends of the non-defective viral genomic RNA. Cloning of this RNA after reverse transcription and polymerase chain reaction amplification was performed in such a way that an exact copy of the defective RNA could be obtained by transcription of the plasmid with T7 RNA polymerase. Sequence analysis of the plasmid allowed further characterization of the defective RNA. It was shown potentially to encode a C-terminally truncated nucleocapsid (NP) protein of 162 amino acids. This truncated NP protein was identified in cells naturally infected with the defective virus preparation. Moreover the protein produced was shown to correspond to the protein synthesized in vitro from the T7 polymerase transcript of the cloned defective genome.
Abstract: The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from cloned genes when the DI RNA was synthesized from plasmid. Limited nucleotide additions or deletions (+7 to -7 nucleotides) in the DI RNA sequence were then made at five different sites, and the different RNA derivatives were tested for their abilities to replicate. Efficient replication was observed only when the total nucleotide number was conserved, regardless of the modifications, or when the addition of a total of 6 nucleotides was made. The replicated RNAs were shown to be properly enveloped into virus particles. It is concluded that, to form a proper template for efficient replication, the Sendai virus RNA must contain a total number of nucleotides which is a multiple of 6. This was interpreted as the need for the nucleocapsid protein to contact exactly 6 nucleotides.
Abstract: The use of a monoclonal antibody defines a subset of Sendai virus M protein representing about 30% of total. This M protein acquires, during the hour following synthesis, an epitope not present on the bulk of M. This epitope maturation is observed in acutely as well as in persistently infected cells. It takes place in vivo in absence of other viral proteins, but it is not observed when the protein is synthesized in a reticulocyte lysate. Epitope maturation does not appear to result from phosphorylation, acylation or disulfide bond formation. If immunofluorescent staining seems to indicate a preferential association of this subset of M protein with nucleocapsids, this is not confirmed by immunogold staining or by nucleocapsid isolation. Incubation of cytoplasmic extracts or of purified M protein in conditions which do not favor M to M protein association results in a relative increase of M protein carrying the maturing epitope. It is concluded that M protein exists in two distinct isoforms.
Abstract: Using the unique sequence organization of copy-back defective interfering (DI) RNAs of paramyxoviruses, Sendai virus (SV), and measles virus copy-back DI RNAs were PCR amplified and cloned, without having to separate them from their helper nondefective genomes. The cloning was designed so that T7 polymerase transcription of the plasmids would generate DI RNAs with the exact 5' and 3' ends. The SV DI clone, transcribed from the plasmid in BHK cells using T7 polymerase produced by a vaccinia virus recombinant, was encapsidated and replicated by the SV-L, P/C, and NP proteins expressed from cloned genes. Such experiments open the possibility of examining the cis-acting sequences involved in viral multiplication directly, without using indirect markers such as CAT activity.
Abstract: A temperature-sensitive mutant of Sendai virus, ts271, when grown at restrictive temperature is known to produce virions lacking integral haemagglutinin-neuraminidase (HN). In this study, it is shown that the transmembrane-cytoplasmic tail of HN is not detected either. This apparent complete lack of HN does not affect budding efficiency.
Abstract: DI viruses and defective viruses generally are widespread in nature. Laboratory studies show that they can sometimes exert powerful disease-modulating effects (either attenuation or intensification of symptoms). Their role in nature remains largely unexplored, despite recent suggestive evidence for their importance in a number of systems.
Abstract: Using Northern blot analysis, we have demonstrated the ability of infectious measles and Sendai virus particles to rescue the intracellular replication of their homologous defective interfering (DI) nucleocapsids up to 3 days and 1 day, respectively, after initial DI infection. The half-life of the paramyxovirus DI nucleocapsids was therefore judged to be similar to that of rhabdoviruses, and to significantly differ from that of orthomyxoviruses. Moreover, we conclude that the intracellular half-life of measles virus DI nucleocapsids makes possible DI replication in the human body after vaccination with a DI-contaminated attenuated live virus, even when this vaccination represents a low multiplicity of infection.
Abstract: From 10-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 30% of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway.
Abstract: Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.
Abstract: The role of glycosylation and of disulfide bonds in the formation of the native structure of the Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins was studied. In contrast to the HN and F0 synthesized in vivo, the proteins made from pSP6 transcripts in reticulocyte lysates, whether glycosylated or not, were not recognized by monoclonal antibodies or polyclonal rabbit sera raised against the native proteins; they efficiently reacted only with rabbit antisera raised against the reduced sodium dodecyl sulfate-denatured proteins. These in vitro-made proteins, however, did not contain disulfide bonds. The proteins made in vivo in the presence of tunicamycin, which were also not recognized by the anti-native protein antibodies, did contain disulfide bonds, but they were mainly incorrect interchain disulfide bonds. Moreover, while F0 acquired proper disulfide bonds as soon as it was synthesized under normal conditions in vivo, the disulfides were formed in HN only after a lag of 10 to 30 min. This lag coincides with the delay observed in HN native structure formation. We therefore conclude that the maturation of the HN and F0 proteins depends on the formation of proper intramolecular disulfide bonds, which in turn depends on the previous addition of high-mannose sugars.
Abstract: The budding efficiency of Sendai virus antigenomes, as well as of defective interfering (DI) nucleocapsids of the deletion and copy-back types, was compared to that of the viral genome during infections of baby hamster kidney (BHK) cells. The antigenomes were shown to bud into virus particles as efficiently as the genomes, arguing for the irrelevance of the nucleocapsid-RNA ends in regulating the efficiency of budding. The DI nucleocapsids, however, were restricted in their budding by factors inversely proportional to their size, arguing for an effect of nucleocapsid size in this process. This restriction in budding, however, appeared to be only expressed under conditions of very efficient DI-RNA replication.
Abstract: Upon infections of BHK cells with a mixture of Sendai standard and defective interfering (DI) viruses (mixed virus infection), viral budding was found to be restricted by factors ranging from 5 to more than 20. The reduced viral budding correlated with a high intracellular M protein turnover. M appeared to be degraded shortly after its synthesis, and seemed not to be able to self-associate in a stable way under the plasma membrane as it did in St virus-infected cells. These data, added to the previous findings that infection with DI particles allowed infected cell survival and favored the cell-surface turnover of the hemagglutinin-neuraminidase protein, led to the hypothesis that DI genomes directly act by preventing the stable formation inside the cells of a viral structure composed of M/HN/nucleocapsids. When involved in this structure M would be protected from degradation and HN would be stably anchored in the plasma membrane. Formation of this structure would be necessary for viral budding and would be damaging for the cells. Comparison with results published by other authors shows that such a model is consistent with other data. It can integrate, as well, data obtained in the analysis of mutant viruses involved in persistence.
Abstract: By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.
