Virus multiplication and induction of apoptosis by Sendai virus: role of the C proteins

Sendai virus (SeV) P gene encodes a nested set of carboxyl-coterminal proteins (C', C, Y1 and Y2), which are referred to collectively as the C proteins. Characterization of the virus multiplication and cellular responses in HEp-2 cells infected with the recombinant SeV which lacks two (C' and C), three (C', C and Y1) or all the four C proteins revealed that all the recombinant viruses can grow in the cells to various extents, depending, apparently, on the number of species expressing C protein. In reverse proportion to the viral growth ability, these viruses induced apoptosis in the infected cells. These results indicate that Y2 protein has an antiapoptotic activity, and suggest that this activity works in an additive manner with the longer C protein(s) (C' and/or C) of SeV in order to suppress virus-induced apoptosis in the SeV-infected cells. Apparently, the antiapoptotic activity of the C proteins supports virus multiplication in the infected cells.     

The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.     

Intracellular processing of the Sendai virus C' protein leads to the generation of a Y protein module: structure-functional implications

The Sendai virus "C-proteins" (C', C, Y1 and Y2) are a nested set of non-structural proteins. The shorter Y proteins arise in vivo both by de novo translation initiation and by proteolytic processing of C'. In this paper, we demonstrate that C' but not C (differing only by 11 N-terminal amino acid) serves as an efficient substrate for intracellular processing. However, processing can be mimicked in vitro by the addition of endopeptidases. Under conditions of limited proteolysis we observed that in a fraction of the C' protein the Y region exists as a proteinase resistant core. This core was conserved in the C protein. We propose that C' functions as a Pro-protein delivering the Y module to a specific intracellular location.     

Proteolytic processing and translation initiation: two independent mechanisms for the expression of the Sendai virus Y proteins

The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.     

Ribosomal initiation at alternate AUGs on the Sendai virus P/C mRNA.

Peptide sera specific for the C, C/C', and P proteins of Sendai virus have been used to confirm that the viral nonstructural proteins originate from internal AUG codons and are translated in a different reading frame from that of the P protein. The C protein undergoes aberrant migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is expressed at higher levels in infected cells than are the P and C' proteins.     

Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon-alpha/beta-mediated responses.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.     

Ribosomal initiation from an ACG codon in the Sendai virus P/C mRNA.

The Sendai virus P/C mRNA expresses the P and C proteins from alternate reading frames. The C reading frame of this mRNA, however, is responsible for three proteins, C', C and Y, none of which appear to be precursors to each other in vivo. Using site-directed and deletion mutagenesis of the P/C gene cloned in SP6 and in vitro translation of the mRNAs, we show that the 5' most proximal initiation codon of the mRNA is an ACG at position 81, responsible for C' synthesis. The succeeding initiation codons, all ATGs, are responsible for the P protein (position 104), the C protein (position 114) and the Y protein(s) (either positions 183 or 201). Examination of the relative molar amounts of the C', P and C proteins found in vivo suggests that an ACG in an otherwise favorable context is almost as efficient for ribosome initiation as an ATG in a less favored context, but only 10-20% as efficient as an ATG in a more favored context. The judicious choice of increasingly more favorable initiation codons in the P/C gene allows multiple proteins to be made from a single mRNA.     

The parainfluenza virus type 1 P/C gene uses a very efficient GUG codon to start its C'' protein.

Parainfluenza virus type 1 (PIV1) and Sendai virus (SEN) are very closely related, but the PIV1 P/C gene does not contain the ACG codon which initiates the SEN C' protein. Nevertheless, a protein corresponding to the PIV1 C' protein was observed both in vivo and in vitro. The initiation site of this protein maps upstream of the PIV1 C protein AUG in a region that does not contain an AUG codon. We have used site-directed mutagenesis to demonstrate that the PIV1 C' protein initiates from a GUG codon, four codons upstream of where the ACG is found in SEN. Remarkably, this GUG appears to initiate in vivo almost as frequently as AUG in the same context. However, whereas GUG permits downstream expression of the P and C proteins, AUG in this context does not. The conservation of an upstream non-AUG initiation codon for C' among PIV1 and SEN suggests that it is important for virus replication, even though some paramyxoviruses express only the C protein and others have no C open reading frame at all.     

Effect of poliovirus superinfection on Sendai virus protein synthesis.

Poliovirus superinfection of Sendai virus-infected cells inhibited the syntheses of the structural P protein and the nonstructural C' and C proteins equally. As the Sendai virus P, C', and C proteins are all translated from the same mRNA by ribosomes which initiate on alternate AUGs and as non-poliovirus protein synthesis is inhibited in poliovirus-infected cells by inactivation of initiation factors responsible for cap group recognition, these results indicate that cap group recognition is important for ribosome initiation on AUGs which are not proximal to the 5' end of the mRNA.     

Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo.

The Sendai virus P/C mRNA contains five ribosomal initiation sites between positions 81 and 201 from the 5' end. One of these sites initiates in the P open reading frame (ORF) (ATG/104), whereas four initiate in the C ORF (ACG/81 and ATGs/114, 183, 201), to give a nested set of C proteins (C', C, Y1, Y2). Introduction of new ATGs or physically breaking the mRNA upstream of these natural sites was used in vitro to prevent ribosomal scanning downstream. The results suggest that a minority of the ribosomes which initiate C (ATG/114) and all of those which initiate Y1 and Y2 (ATGs/183 and 201) do so by a scanning independent mechanism. When the leaky ACG/81 site is changed to a non-leaky ATG site in in vivo experiments, ribosomal initiation at Y is again not diminished, whereas that at C as well as at P becomes undetectable. Ribosomal initiation at Y appears to be scanning independent in vitro and in vivo. That at C is partly independent in vitro, but completely dependent in vivo. These results are discussed in terms of a model of internal initiation at Y.     

Translational modulation in vitro of a eukaryotic viral mRNA encoding overlapping genes: ribosome scanning and potential roles of conformational changes in the P/C mRNA of Sendai virus

Expression of proteins from three overlapping genes in a single mRNA species of Sendai virus was modulated in a cell-free rabbit reticulocyte translation system. Hybrid-arrested translation by oligodeoxynucleotides complementary to specific regions of the mRNA that specifies the viral P, C, and C' proteins demonstrated that ribosomes scan the RNA from its 5' end to find initiation codons, and suggested that the secondary structure of the mRNA influences the selection of alternative initiation codons. Translational modulation of P, C, and C' proteins by Mg++ and spermidine indicated that RNA folding is involved in this selection process.     

Versatility of the accessory C proteins of Sendai virus: contribution to virus assembly as an additional role

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.     

Identification of a cis-acting element required for shunt-mediated translational initiation of the Sendai virus Y proteins

Shunting is a mechanism that permits translational initiation at internal codons positioned in proximity to a ribosome acceptor sequence. Sendai virus exploits shunting to express a series of proteins that initiate at the fourth and fifth start sites on the P/C mRNA (namely, the Y1 and Y2 proteins, respectively). Shunt-mediated initiation at these sites is codon independent. In an attempt to characterise the acceptor site, an extensive deletion analysis was performed spanning the entire C ORF. Only mutants flanking the Y1/Y2 start sites exhibited altered shunt phenotypes. Some of these significantly enhanced shunting efficiency to the point where the Y1/Y2 proteins were the major translational products of the mRNA. Additionally, removal of a short region just downstream of the Y2 start codon (referred to as Δ10) ablated all Y protein initiation via shunting but had no effect on Y expression when the AUG codons were viewed by a scanning ribosome. Point mutations introduced into this Δ10 sequence severely perturbed shunt-mediated initiation. We also provide evidence that changes in this region of the P/C mRNA may be used to modulate Y protein expression levels in different viral strains.     

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.     

ACG, the initiator codon for a Sendai virus protein

Deletion and site-directed point mutants of the polycistronic P/C mRNA of Sendai virus revealed that one of the nonstructural proteins of this virus, the C' protein, initiates from an ACG codon. This ACG codon occurs in an optimum sequence context and precedes the first AUG of the P/C mRNA. The results presented in this communication are consistent with the concept that the ribosomes scan the P/C mRNA to initiate several proteins from its different initiator codons. The arrangement of several weak initiator codons in tandem in an mRNA, i.e. non-AUG in optimum sequence context and AUG in suboptimum sequence context, may represent an alternate means to regulate gene expression in eukaryotes and their viruses.     

Factor determining the antigenic type of interferons produced in human lymphoblastoid cell lines

The factor that determines the antigenic type of IFN produced in human lymphoblastoid cell lines was examined using live Sendai virus, ultraviolet (UV)-irradiated virus, HANA spikes exposed on L cells persistently infected with Sendai virus (L-HVJ) and poly-inosinic acid poly-cytidylic acid (poly I: C). When Sendai virus was irradiated with UV-light for 300 sec, its abilities to infect chicken eggs and induce IFN were diminished, but its HA activity was unaffected. HANA spikes exposed on L-HVJ could not induce IFN in human lymphoblastoid cell lines, although they induced IFN in mouse spleen cells. These results suggest that the induction of IFN in human lymphoblastoid cells is closely related to viral nucleic acid. Poly I: C also induced IFN in some human lymphoblastoid cell lines in which IFN production is induced by Sendai virus. The antigenic types of IFN induced by poly I: C were the same as those induced by Sendai virus. These results suggest that the antigenic type of IFN produced depends on the nature of the IFN producer cells rather than on the kind of IFN inducer.     

Passage of a Sendai Virus Recombinant in Embryonated Chicken Eggs Leads to Markedly Rapid Accumulation of U-to-C Transitions in a Limited Region of the Viral Genome

The P gene of paramyxoviruses is unique in producing not only P but also “accessory” C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C’, C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported.