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.     

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.     

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.     

Expression of five proteins from the Sendai virus P/C mRNA in infected cells.

The polycistronic P/C mRNA of Sendai virus is translated under cell-free conditions into five proteins (P, C', C, Y1, and Y2) from overlapping reading frames. In this study, we showed that in addition to the P, C', and C proteins, Y1 and Y2 were expressed by six different Sendai virus strains in infected cells. The Y proteins exhibited strain-specific variation in their gel mobility which corresponds to the variation seen in the cognate C proteins. While the relative levels of the P, C', and C proteins were consistent among various cell lines, the levels of Y1 and Y2 proteins varied among the cell lines used for viral infection.     

Endocytosis plays a critical role in proteolytic processing of the Hendra virus fusion protein

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.     

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.     

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.     

The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation

Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.     

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.     

Efficient RNA 2'-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C'/D' RNPs

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.     

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.     

The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology

TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.     

Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): a Y210C mutation causes either altered protein handling or altered protein function of N-acetylgalactosamine 4-sulfatase at multiple points in the vacuolar network

The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

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.