ABSTRACT
The RNA dependent RNA polymerase of Sendai virus consists of a complex of the large (L) and phosphoprotein (P) subunits where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. We previously showed that the L protein forms an oligomer [Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245] and mapped the L oligomerization domain between amino acids 1 and 174 of the protein [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536]. An internal deletion encompassing amino acids 20 to 178 of the L protein lost polymerase activity but still formed an L-L oligomer. The first 25 amino acids of paramyxovirus L proteins are highly conserved and site-directed mutagenesis within this region eliminated the biological activity of the L protein but did not have any effect on P-L or L-L interactions. Moreover deletion of amino acids 2-18 in L abolished biological activity, but again the L-L binding was normal demonstrating that the oligomerization domain of L protein resides in two N-terminal regions of the protein. Therefore, sequences between both aa 2-19 and aa 20-178 can independently mediate Sendai L oligomerization, however, both are required for the activity of the protein.
Subject(s)
Protein Subunits/chemistry , Protein Subunits/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Sendai virus/enzymology , Binding Sites , Cell Line, Tumor , Humans , Protein Binding , Protein Structure, Quaternary , Sequence DeletionABSTRACT
While the interferon (IFN)-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR is reported to initiate apoptosis in some instances, the mechanism by which diverse stress stimuli activate PKR remains unknown. Now we report that RAX, the only known cellular activator for PKR, initiates PKR activation in response to a broad range of stresses including serum deprivation, cytotoxic cytokine or chemotherapy treatment, or viral infection. Thus, knock-down of RAX expression by 80% using small interfering RNA (siRNA) prevents IFNgamma/tumor necrosis factor alpha (TNFalpha)-induced PKR activation and eIF2alpha phosphorylation, IkappaB degradation, IRF-1 expression, and STAT1 phosphorylation, resulting in enhanced murine embryonic fibroblast (MEF) cell survival. In contrast, expression of exogenous RAX, but not of the nonphosphorylatable, dominant-negative RAX(S18A) mutant, sensitizes cells to IFNgamma/TNFalpha, mitomycin C (MMC), or serum deprivation in association with increased PKR activity and apoptosis. Furthermore, RAX(S18A) expression in Fanconi anemia complementation group C-null MEF cells not only prevents PKR activation but also blocks hypersensitivity to IFNgamma/TNFalpha or mitomycin C that results in enhanced apoptosis. In addition, reduced RAX expression facilitates productive viral infection with vesicular stomatitis virus (VSV) and promotes anchorage-independent colony growth of MEF cells. Collectively, these data indicate that RAX may function as a negative regulator of growth that is required to activate PKR in response to a broad range of apoptosis-inducing stress.
Subject(s)
Eye Proteins/physiology , Homeodomain Proteins/physiology , RNA-Binding Proteins/physiology , Stress, Physiological/metabolism , Transcription Factors/physiology , eIF-2 Kinase/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line , Cell Proliferation , Cytokines/pharmacology , Eye Proteins/genetics , Fibroblasts/cytology , Homeodomain Proteins/genetics , Humans , Inflammation , Mice , Phlebotomy , RNA, Small Interfering/pharmacology , RNA-Binding Proteins/genetics , Transcription Factors/genetics , Virus Diseases/etiologyABSTRACT
The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.
Subject(s)
DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , RNA Caps/genetics , RNA, Messenger/genetics , Vesicular stomatitis Indiana virus/enzymology , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Conserved Sequence , Cricetinae , Humans , Methylation , Molecular Sequence Data , RNA, Viral/genetics , Rhabdoviridae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Viral ProteinsABSTRACT
The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5'-cap structures methylated at the guanine-N7 and 2'-O-adenosine positions (7mGpppA(m)). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34 degrees C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40 degrees C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5'-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.
Subject(s)
Amino Acid Substitution , RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Vesicular stomatitis Indiana virus/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Humans , Methylation , Molecular Sequence Data , Mutation , RNA-Dependent RNA Polymerase/metabolism , Recombination, Genetic , Temperature , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Viral Proteins/metabolism , Virus ReplicationABSTRACT
Measles virus encodes an RNA-dependent RNA polymerase composed of the L and P proteins. Recent studies have shown that the L proteins of both Sendai virus and parainfluenza virus 3 form an L-L complex [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very Nterminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536.; Smallwood, S., Moyer, S.A., 2004. The L polymerase protein of parainfluenza virus 3 forms anoligomer and can interact with the heterologous Sendai virus L, P and C proteins. Virology 318, 439-450.; Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245.]. Using differentially tagged L proteins, we show here that measles L also forms an oligomer and the L-L binding site resides in the N-terminal 408 amino acids overlapping the P binding site in the same region of L. To identify amino acids important for binding P and L, site-directed mutagenesis of the L-408 protein was performed. Seven of twelve mutants in L-408 were unable to form a complex with measles P while the remainder did bind at least some P. In contrast, all of the mutants retained the ability to form the L-L complex, so different amino acids are involved in the L and P binding sites on L. Four of the 408 mutations defective in P binding were inserted into the full-length measles L protein and all retained L-L complex formation, but did not bind P. Full-length L mutants that did not bind P were also inactive in viral RNA synthesis, showing a direct correlation between P-L complex formation and activity.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , Measles virus/enzymology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Binding Sites , Cell Line, Tumor , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Viral , Humans , Measles virus/genetics , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Viral Proteins/geneticsABSTRACT
To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.
Subject(s)
Nucleocapsid/chemistry , Nucleocapsid/metabolism , Nucleoproteins/chemistry , Nucleoproteins/metabolism , Phosphoproteins/metabolism , Sendai virus/metabolism , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Humans , Macromolecular Substances , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleocapsid/genetics , Nucleocapsid Proteins , Nucleoproteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Sendai virus/genetics , Sequence Deletion , Viral Core Proteins/genetics , Virus ReplicationABSTRACT
Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.
Subject(s)
Down-Regulation , Interferons/antagonists & inhibitors , RNA, Viral/biosynthesis , RNA, Viral/drug effects , Sendai virus/pathogenicity , Viral Proteins/chemistry , Amino Acid Substitution , Antiviral Agents , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Interferon-alpha/antagonists & inhibitors , Interferon-alpha/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/metabolism , Interferons/metabolism , Phosphorylation , STAT1 Transcription Factor , STAT2 Transcription Factor , Trans-Activators/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/pharmacologyABSTRACT
We recently showed that the L protein of Sendai virus is present as an oligomer in the active P-L polymerase complex [Smallwood et al., Virology 304 (2002) 235]. We now demonstrate using two different epitope tags that the L protein of a second respirovirus, human parainfluenza type 3 virus (PIV3), also forms an L-L complex. L oligomerization requires the coexpression of the differentially epitope tagged L proteins. By exploiting a series of C-terminal truncations the L-L binding site maps to the N-terminal half of L. There is some complex formation between the heterologous PIV3 and Sendai L and P proteins; however, the heterologous L protein does not function in transcription of either the PIV3 or Sendai template. The PIV3 C protein binds PIV3 L and inhibits RNA synthesis in vitro and in vivo. Significant homology exists between the C proteins of PIV3 and Sendai and complex formation occurs between the PIV3 and Sendai heterologous C and L proteins. In addition, the heterologous C proteins can inhibit transcription at approximately 50% of the level of the homologous protein. These data suggest that while the C proteins may be functionally somewhat interchangeable, the L and P proteins are specific for each virus.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Phosphoproteins/metabolism , Viral Proteins/metabolism , Animals , Cell Line , Dimerization , Humans , Mutation , Parainfluenza Virus 3, Human/metabolism , Protein Binding , RNA, Viral/metabolism , Sendai virus/metabolism , Transcription, Genetic , Viral Proteins/geneticsABSTRACT
The Sendai virus RNA-dependent RNA polymerase is composed of the L and P proteins. We previously showed that the L protein gives intragenic complementation and forms an oligomer where the L-L interaction site mapped to the N-terminal half of the protein (S. Smallwood et al., 2002, Virology, 00, 000-000). We now show that L oligomerization does not depend on P protein and progressively smaller N-terminal fragments of L from amino acids (aa) 1-1146 through aa 1-174 all bind wild-type L. C-terminal truncations up to aa 424, which bind L, can complement the transcription defect in an L mutant altered at aa 379, although these L truncation mutants do not bind P. The fragment of L comprising aa 1-895, furthermore, acts as a dominant-negative mutant to inhibit transcription of wild-type L. N-terminal deletions of aa 1-189 and aa 1-734 have lost the ability to form the L-L complex as well as the L-P complex, although they still bind C protein. These data are consistent with the L-L interaction site residing in aa 1-174. Site-directed mutations in the N-terminal 347 aa, of L which abolish P binding, do not affect L-L complex formation, so while the L and P binding sites on L are overlapping they are mediated by different amino acids. The N-terminal portions of L with aa 1-424, aa 1-381, and to a lesser extent aa 1-174, can complement the transcription defect in an L mutant altered at aa 77-81, showing their L-L interaction is functional.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , RNA-Dependent RNA Polymerase/metabolism , Sendai virus/metabolism , Viral Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Dimerization , Humans , Mutation , Phosphoproteins/metabolism , Protein Binding , RNA-Dependent RNA Polymerase/chemistry , Transcription, Genetic , Tumor Cells, Cultured , Viral Proteins/geneticsABSTRACT
The RNA-dependent RNA polymerase of Sendai virus consists of two subunits, the L and P proteins, where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of a variety of negative-stranded RNA viruses revealed six regions of good conservation, designated domains I-VI, which are thought to correspond to functional domains of the protein. Analysis of a number of site-directed mutants within the six domains of L allowed us to conclude that the activities of the polymerase are not simply compartmentalized and that each domain contributes to multiple steps in viral RNA synthesis. Nevertheless these domains can function in trans since we demonstrate here that intragenic complementation between pairs of coexpressed inactive L mutants can restore viral RNA synthesis on an added template. Although intragenic complementation is typically very inefficient, complementation to restore leader RNA synthesis was surprisingly very efficient for some pairs and complementation of mRNA synthesis and genome replication was less, but still significant. Complementation occurred with L mutants in five of the six domains, the exception being a domain III mutant, and required the cotranslation of the two L mutants. C-terminal truncations deleting up to half of L were capable of restoring transcription of an inactive domain I L mutant at amino acid 379. Oligomerization of L in the polymerase complex was demonstrated directly by the co-immunoprecipitation of differentially epitope-tagged full-length and truncated L proteins. These data are consistent with L protein being an oligomer with multiple independent domains each of which exhibits several functions.
Subject(s)
RNA-Dependent RNA Polymerase/chemistry , Sendai virus/genetics , Humans , Protein Biosynthesis , Protein Subunits , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/physiology , Sendai virus/enzymology , Transcription, Genetic , Tumor Cells, Cultured , Virus ReplicationABSTRACT
The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , RNA, Viral/biosynthesis , Sendai virus/enzymology , Viral Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Humans , Molecular Sequence Data , Mutation , Nucleocapsid/metabolism , Phosphoproteins/metabolism , Protein Structure, Tertiary , RNA, Messenger/biosynthesis , Sendai virus/genetics , Sendai virus/physiology , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Viral Proteins/chemistry , Viral Proteins/genetics , Virus ReplicationABSTRACT
Sendai virus encodes an RNA-dependent RNA polymerase which is composed of the L and P proteins. Site-directed mutagenesis of the N terminus of L has identified amino acids important for binding P. Seven of nine mutants in amino acids 1 to 350 of Sendai L lost the ability to bind to Sendai P, although they were still able to bind the viral C protein. Loss of P binding correlated with the loss of all RNA synthesis activities. Two L mutants gave limited P-L complex formation and limited viral transcription and replication.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Phosphoproteins/metabolism , Sendai virus/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Substitution , Binding Sites , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Mutagenesis, Site-Directed , Phosphoproteins/chemistry , Phosphoproteins/genetics , RNA, Viral/metabolism , Sendai virus/genetics , Transcription, Genetic , Viral Proteins/geneticsABSTRACT
The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others--JT4, JT6, and JT9--were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP(0) binding and abolished the reconstitution of replication from separate P-L and NP(0)-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP(0)-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.