ABSTRACT
The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.
Subject(s)
Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA, Viral/biosynthesis , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/metabolism , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Amino Acid Motifs , Humans , Mass Spectrometry , Phosphoproteins/genetics , Phosphorylation , Protein Processing, Post-Translational , RNA, Viral/genetics , Serine/genetics , Serine/metabolism , Tyrosine/genetics , Tyrosine/metabolism , Vesicular stomatitis Indiana virus/chemistry , Vesicular stomatitis Indiana virus/genetics , Viral Structural Proteins/genetics , Virus ReplicationABSTRACT
The active template for RNA synthesis for vesicular stomatitis virus (VSV) and other negative-strand viruses is the RNA genome in association with the nucleocapsid (N) protein. The N protein molecules sequester the genomic RNA and are linked together by a network of noncovalent interactions. We previously demonstrated that mutations predicted to weaken interactions between adjacent N protein molecules altered the levels of RNA synthesis directed from subgenomic ribonucleoprotein (RNP) templates. To determine if these mutations affect virus replication, recombinant viruses containing single-amino-acid substitutions in the N protein were recovered. Four mutations altered transcription and genome replication levels, perturbed viral protein synthesis, and inhibited virus replication. Selective pressure for improved virus replication was applied by eight sequential passages. After five passages, virus replication improved and RNA synthesis recovered concomitantly with the restoration of the protein molar ratios to near-wild-type levels. Genome sequences were compared before and after passage to determine whether compensatory mutations were selected and to potentially identify interactions between N protein molecules or between the RNP template and the viral polymerase. Improved virus replication correlated with the selection of additional mutations located in cis-acting transcriptional regulatory sequences at the gene junctions of the genome rather than in coding sequences, with one exception. The engineered N gene mutations perturbed mRNA and protein expression levels, but the selection of modified transcriptional regulatory sequences with passage facilitated the restoration of wild-type protein expression by modulating transcription levels, reflecting the adaptability and versatility of gene regulation by transcriptional control.
Subject(s)
Mutation , Nucleocapsid Proteins/genetics , Regulatory Elements, Transcriptional/genetics , Vesicular stomatitis Indiana virus/genetics , Virus Replication , Amino Acid Substitution , Animals , Base Sequence , Cell Line , Cricetinae , Gene Expression Regulation, Viral , Genes, Viral , Genome, Viral , Nucleocapsid Proteins/chemistry , Protein Biosynthesis , RNA, Viral/biosynthesis , RNA, Viral/genetics , Sequence Analysis, RNA , Transcription, Genetic , Vesicular Stomatitis/virologyABSTRACT
Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.
ABSTRACT
Commentary on Ge, P.; Tsao, J.; Schein, S.; Green, T.J.; Luo, M.; Zhou, Z.H. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science2010, 327, 689-693.
ABSTRACT
Vesicular stomatitis virus (VSV) genomic RNA encapsidated by the nucleocapsid (N) protein is the template for transcription and replication by the viral polymerase. We analyzed the 2.9-A structure of the VSV N protein bound to RNA (T. J. Green, X. Zhang, G. W. Wertz, and M. Luo, Science 313:357-360, 2006) and identified amino acid residues with the potential to interact with RNA via hydrogen bonds. The contributions of these interactions to N protein function were investigated by individually substituting the residues with alanine and assaying the effect of these mutations on N protein expression, on the ability of the N protein to interact with the phosphoprotein (P), and on its ability to encapsidate RNA and generate templates that can support transcription and RNA replication. These studies identified individual amino acids critical for N protein function. Nine nucleotides are associated with each N monomer and contorted into two quasi-helices within the N protein RNA binding cavity. We found that N protein residues that formed hydrogen bond contacts with the nucleotides in quasi-helix 2 were critical to the encapsidation of RNA and the production of templates that can support RNA synthesis. Individual hydrogen bond interactions between the N protein and the nucleotides of quasi-helix 1 were not essential for ribonucleoprotein (RNP) template function. Residue R143 forms a hydrogen bond with nucleotide 9, the nucleotide that extends between N monomers. R143A mutant N protein failed to encapsidate RNA and to support RNA synthesis and suppressed wild-type N protein function. These studies show a direct correlation between viral RNA synthesis and N protein residues structurally positioned to interact with RNA.
Subject(s)
Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/physiology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Vesiculovirus/genetics , Vesiculovirus/physiology , Amino Acid Substitution , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Cricetinae , DNA Primers/genetics , Genome, Viral , Hydrogen Bonding , Macromolecular Substances , Models, Molecular , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Nucleocapsid Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/physiology , Protein Conformation , RNA, Viral/chemistry , Transfection , Viral Structural Proteins/chemistry , Viral Structural Proteins/genetics , Viral Structural Proteins/physiology , Virus AssemblyABSTRACT
The 2.9-A structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template. We investigated the roles of individual amino acid residues in the C-terminal loop, involved in long-range interactions between N protein monomers, in forming functional ribonucleoprotein (RNP) templates. The effects of specific N protein mutations on its expression, interaction with the phosphoprotein, and formation of RNP templates that supported viral RNA replication and transcription were examined. Mutations introduced into the C-terminal loop, predicted to break contact with other residues in the loop, caused up to 10-fold increases in RNA replication without an equivalent stimulation of transcription. Mutation F348A, predicted to break contact between the C-terminal loop and the N-terminal arm, formed templates that supported wild-type levels of RNA replication but almost no transcription. These data show that mutations in the C-terminal loop of the N protein can disparately affect RNA replication and transcription, indicating that the N protein plays a role in modulating RNP template function beyond its structural role in RNA encapsidation.
Subject(s)
Nucleocapsid Proteins/genetics , Reverse Transcription/genetics , Vesiculovirus/genetics , Virus Replication/genetics , Blotting, Western , Gene Expression Regulation, Viral/genetics , Immunoprecipitation , Mutation/genetics , Nucleocapsid Proteins/chemistry , RNA, Viral/biosynthesis , RNA, Viral/genetics , Ribonucleases/metabolismABSTRACT
To investigate the polymerase components selectively involved in transcription versus replication of vesicular stomatitis virus (VSV), we sequenced the polymerase gene of a conditionally RNA defective, temperature sensitive VSV: ts(G)114, which has a phenotype upon shift from permissive to non-permissive temperature of shut-down of mRNA transcription and unaffected genome replication. Sequence analysis of the ts(G)114 L gene identified three altered amino acid residues in the L protein. These three changes were specifically engineered individually and in combinations into a functional cDNA clone encoding the VSV genome and tested for association with the temperature sensitive and RNA defective phenotypes in the background of recovered engineered viruses. The data presented in this study show a specific amino acid substitution in domain II of the VSV L protein that significantly affects total RNA synthesis, but when in combination with two additional amino acid substitutions identified in the ts(G)114 L protein, leads to a specific reduction in mRNA transcription, but not replication.
Subject(s)
Gene Expression Regulation, Viral , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , Temperature , Transcription, Genetic , Vesiculovirus/physiology , Viral Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Vero Cells , Vesiculovirus/genetics , Vesiculovirus/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Virion/metabolism , Virus Replication/geneticsABSTRACT
There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3' end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5' cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM. To investigate this unusual phenotype, we assayed the effects of SAH on transcription using a panel of recombinant viruses that contained mutations in domain VI of the VSV L protein. The L proteins we investigated displayed a range of 5' cap methyltransferase activities. In the present study, we show that the ability of the VSV L protein to catalyze methyl transfer correlates with its sensitivity to SAH with respect to polyadenylation, thereby indicating an intriguing connection between 5' and 3' end mRNA modifications. We also identified an L protein mutant that hyperpolyadenylates mRNA irrespective of the presence or absence of exogenous SAH. Further, the data presented here show that the wild-type L protein hyperpolyadenylates a percentage of VSV mRNAs in infected cells as well as in vitro.
Subject(s)
Adenosine/genetics , Adenosine/metabolism , Polymers/metabolism , Protein Methyltransferases/metabolism , RNA-Dependent RNA Polymerase/metabolism , S-Adenosylhomocysteine/pharmacology , Vesiculovirus/drug effects , Vesiculovirus/metabolism , Viral Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Cricetinae , Methylation , Models, Molecular , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Substrate Specificity , Transcription, Genetic/genetics , Vesiculovirus/genetics , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The large (L) proteins of non-segmented negative stranded (NNS) RNA viruses contain the core RNA dependent RNA polymerase activity for RNA replication and transcription as well as the activities for polyadenylating and capping the mRNA transcripts and for methylating the cap structures. There is currently no structural information available for these large multi-functional proteins. Phylogenetic analyses have led to the division of the L protein primary structure into six functional domains of high conservation that are linked by variable regions. The studies in this report investigate the role of specific amino acids within domain VI of the VSV L protein, which contains a 2'-O-ribose methyltransferase (MTase) domain. We generated a structural homology model of residues 1644-1842 within domain VI based on the crystal structure determined for the known 2'-O-ribose MTase of E. coli, RrmJ. The information generated by this homology model directed us to residues structurally important for MTase activity and SAM binding. Selected residues were analyzed by site-specific mutagenesis and the mutant L proteins were assayed for their effects on RNA synthesis and cap methylation. The goal of this study was to functionally test the model in order to gain insight into the structural constraints of this region of the L protein. The data presented here revealed specific mutations that affect transcription, replication, and 5' cap methylation, many of which resulted in polymerases temperature sensitive for RNA synthesis.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Methyltransferases/chemistry , Methyltransferases/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Amino Acid Substitution/genetics , Archaeal Proteins/metabolism , Cell Cycle Proteins/chemistry , Methylation , Methyltransferases/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Structure, Tertiary , RNA Caps/metabolism , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolismABSTRACT
The heptauridine tract at each gene end and intergenic region (IGR) at the gene junctions of vesicular stomatitis virus (VSV) have effects on synthesis of the downstream mRNA, independent of their respective roles in termination of the upstream mRNA. To investigate the role of the U tract and the IGR in downstream gene transcription, we altered the N/P gene junction of infectious VSV such that transcription levels would be affected and result in altered molar ratios of the N and P proteins, which are critical for optimal viral RNA replication. The changes included extended IGRs between the N and P genes and shortening the length of the heptauridine tract upstream of the P gene start. Viruses having various combinations of these changes were recovered from cDNA and selective pressure for efficient viral replication was applied by sequential passage in cell culture. The replicative ability and sequence at the altered intergenic junctions were monitored throughout the passages to compare the effects of the changes at the IGR and U tract. VSV variants with wild-type U tracts upstream of the P gene replicated to levels similar to wt VSV. Variants with shortened U tracts were reduced in their ability to replicate. With passage, populations emerged that replicated to higher levels. Sequence analysis revealed that mutations had been selected for in these populations that increased the length of the U tract. This correlated with an increase in abundance of P mRNA and protein to provide improved N:P protein molar ratios. Extended IGRs resulted in decreased downstream transcription but the effect was not as extensive as that caused by shortened U tracts. Extended IGRs were not selected against in 5 passages. Our results indicate that the size of the upstream gene end U tract is an important determinant of efficient downstream gene transcription in infectious virus.
Subject(s)
DNA, Intergenic/genetics , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , Transcription, Genetic , Vesiculovirus/physiology , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Cricetinae , Mutagenesis, Insertional , Selection, Genetic , Sequence Deletion , Vesiculovirus/genetics , Viral Plaque Assay , Viral Proteins/biosynthesisABSTRACT
Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.
Subject(s)
Cell Polarity , Epithelial Cells/metabolism , Respiratory Syncytial Virus, Human/physiology , Animals , Carcinoma, Hepatocellular/pathology , Cell Line , Chlorocebus aethiops , DNA, Complementary , Dogs , Gene Deletion , Genes, Reporter , Genetic Engineering , Glycoproteins/analysis , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Kidney/cytology , Liver Neoplasms/pathology , Open Reading Frames , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/pathogenicity , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bi-cistronic replicons were constructed containing an mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed.
Subject(s)
DNA, Intergenic/genetics , RNA, Messenger/metabolism , RNA-Dependent RNA Polymerase/metabolism , Transcription Initiation Site , Transcription, Genetic , Vesicular stomatitis Indiana virus/metabolism , Animals , Base Sequence , Cell Line , Cricetinae , Molecular Sequence Data , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , Transfection , Vesicular stomatitis Indiana virus/enzymology , Vesicular stomatitis Indiana virus/geneticsABSTRACT
Current efforts to develop a vaccine against human respiratory syncytial virus (HRSV) are focused on live attenuated strains. However, the unstable nature of HRSV is a major challenge for the preparation, storage and distribution of live vaccine candidates. We report here that the stability of HRSV can be improved by incorporation of the GP64 glycoprotein from baculovirus Autographa californica multiple nucleopolyhedrovirus. GP64 was incorporated in place of or in addition to the homologous HRSV glycoproteins and was either expressed from the HRSV genome or provided by propagating the virus in a Vero cell line constitutively expressing GP64 (Vbac cells). The infectivity of the different virus stocks was monitored after storage at 4 degrees, 22 degrees or 37 degrees C, over a period of 8 weeks. The results showed that the infectivity of HRSV could be stabilized by up to 10,000-fold by the GP64 protein, when stored at 22 degrees C for 6 weeks. This approach for stabilizing live HRSV may be important for vaccine development and may also prove useful for stabilizing other enveloped viruses.
Subject(s)
Cell Adhesion Molecules/genetics , Membrane Glycoproteins/genetics , Respiratory Syncytial Virus, Human/genetics , Viral Proteins/genetics , Animals , Baculoviridae/genetics , Baculoviridae/immunology , Blotting, Western , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Genetic Engineering , Glycoproteins/chemistry , Glycoproteins/genetics , Humans , Respiratory Syncytial Virus, Human/chemistry , Respiratory Syncytial Virus, Human/pathogenicity , Temperature , Vero CellsABSTRACT
The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.
Subject(s)
Respiratory Syncytial Virus, Human/physiology , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/physiology , Amino Acid Sequence , Animals , Cell Line , Chlorocebus aethiops , DNA, Complementary/genetics , DNA, Viral/genetics , Humans , Membrane Fusion , Membrane Microdomains/metabolism , Membrane Microdomains/virology , Molecular Sequence Data , Mutagenesis , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/pathogenicity , Vero Cells , Viral Fusion Proteins/genetics , Virus ReplicationABSTRACT
Vesicular stomatitis virus is a negative-stranded RNA virus. Its nucleoprotein (N) binds the viral genomic RNA and is involved in multiple functions including transcription, replication, and assembly. We have determined a 2.9 angstrom structure of a complex containing 10 molecules of the N protein and 90 bases of RNA. The RNA is tightly sequestered in a cavity at the interface between two lobes of the N protein. This serves to protect the RNA in the absence of polynucleotide synthesis. For the RNA to be accessed, some conformational change in the N protein should be necessary.
Subject(s)
Nucleocapsid Proteins/chemistry , RNA, Viral/chemistry , Ribonucleoproteins/chemistry , Vesicular stomatitis Indiana virus/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Nucleocapsid Proteins/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Viral/metabolism , Sequence AlignmentABSTRACT
Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3' ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5' terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3'-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5' non-translated region (NTR) of the S segment. A 6 nt region (3'-GUCGAC-5') within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3'-UGUCG-5', that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5' NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.
Subject(s)
Bunyamwera virus/genetics , Genome, Viral , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic , 3' Untranslated Regions/chemistry , 3' Untranslated Regions/genetics , 5' Untranslated Regions/chemistry , 5' Untranslated Regions/genetics , Bunyamwera virus/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Virus Replication/geneticsABSTRACT
Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of RNA viruses. BUNV genomic strands are templates for both replication and transcription, whereas the antigenomic RNAs serve only as templates for replication. By mutagenesis of model templates, we showed that the BUNV transcription promoter comprises elements within both the 3' and the 5' nontranslated regions. Using this information, we designed a model ambisense BUNV segment that transcribed BUNV S mRNA from the genomic strand and green fluorescent protein (GFP) mRNA from the antigenome. Demonstration of GFP expression showed that this ambisense strategy represents a viable approach for generating BUNV segments able to express additional proteins.
Subject(s)
3' Untranslated Regions , 5' Untranslated Regions , Bunyamwera virus/genetics , RNA, Viral/genetics , Transcription, Genetic , Base Sequence , Bunyamwera virus/physiology , Genes, Reporter , Green Fluorescent Proteins/analysis , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolismABSTRACT
We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ. We show that the capacities of viruses with either the GNJ or GI glycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJ could infect cells at acidic pHs, while the infectivity of VSIV-GI was severely reduced. VSIV-GNJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GI by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-GI variant (VSIV-6.8) that was similar to VSIV-GNJ regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-GI full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.
Subject(s)
Amino Acids/physiology , Glycoproteins/genetics , Glycoproteins/physiology , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/physiology , Vesiculovirus , Viral Envelope Proteins/physiology , Amino Acid Substitution , Amino Acids/genetics , Animals , Cells, Cultured , Chlorocebus aethiops , DNA Mutational Analysis , Glycoproteins/chemistry , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Selection, Genetic , Serotyping , Vero Cells , Vesicular stomatitis Indiana virus/classification , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Plaque AssayABSTRACT
Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3' and 5' nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable. Here, we investigate the basis for the fundamentally different signaling abilities of genomic and antigenomic strands. We show that the identity of only nucleotide position 9 within the genomic 3' NTR is critical for the different RNA synthesis characteristics of genomic and antigenomic strands, thus identifying this nucleotide as an essential component of the transcription promoter. This nucleotide is distinctive, as it interrupts an unbroken run of conserved complementary nucleotides within the 3' and 5' NTRs of all three segments. Our results show that the conserved mismatched arrangement of this nucleotide plays no detectable role in signaling transcription. Instead, we show that the transcription-signaling ability of this position is entirely dependent on its nucleotide identity. We further show that while a U residue at 3' position 9 is strongly preferred for transcription activity in the context of the genomic promoter, it does not signal transcription in the context of the antigenomic promoter. Therefore, our results show that the identity of 3' position 9 is crucial for signaling BUNV transcription; however, it is not the sole determinant.
Subject(s)
3' Untranslated Regions , 5' Untranslated Regions , Base Pair Mismatch , Bunyamwera virus/physiology , Transcription, Genetic , Virus Replication , Bunyamwera virus/genetics , Nucleic Acid Conformation , Nucleotides/genetics , Nucleotides/physiology , Promoter Regions, Genetic , RNA, Viral/genetics , RNA, Viral/metabolism , Regulatory Sequences, Nucleic Acid , Templates, GeneticABSTRACT
The Respiratory Syncytial Virus 2003 symposium took place from 8th-11th November 2003 in Stone Mountain, Georgia, and brought together more than 200 international investigators engaged in RSV research. RSV biology, pathogenesis, and clinical data, as well as RSV vaccines and antivirals, were addressed in the meeting, and this review will aim to briefly summarize and discuss the implications of new findings. The meeting also served as the inauguration of the Robert M. Chanock Award for lifetime achievement in RSV research, an award named in honor of the person who started the field of RSV research by recovering the first human RS virus from infants with severe bronchiolitis in 1956.