scholarly journals Identification of Mutations Causing Temperature-Sensitive Defects in Semliki Forest Virus RNA Synthesis

2006 ◽  
Vol 80 (6) ◽  
pp. 3108-3111 ◽  
Author(s):  
Valeria Lulla ◽  
Andres Merits ◽  
Peter Sarin ◽  
Leevi Kääriäinen ◽  
Sirkka Keränen ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Nonstructural Protein ◽  
Prototype Strain ◽  
Temperature Sensitive ◽  
Helicase Domain ◽  
Coding Region ◽  
Protein Coding ◽  
Subgenomic Rna ◽  
The Individual

ABSTRACT We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.

scholarly journals nsP4 is a major determinant of alphavirus replicase activity and template selectivity

2021 ◽  
Author(s):  
Laura Sandra Lello ◽  
Koen Bartholomeeusen ◽  
Sainan Wang ◽  
Sandra Coppens ◽  
Rennos Fragkoudis ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Subgenomic Rna ◽  
Positive Strand ◽  
Novel Approach ◽  
Positive Strand Rna ◽  
Chimeric Viruses

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the non-structural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1-nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis -active sequences. Here we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123 while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. Importance. A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

scholarly journals The Red Clover Necrotic Mosaic Virus RNA2 trans-Activator Is Also a cis-Acting RNA2 Replication Element

2005 ◽  
Vol 79 (2) ◽  
pp. 978-986 ◽  
Author(s):  
Masahiro Tatsuta ◽  
Hiroyuki Mizumoto ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Rna Synthesis ◽  
Red Clover ◽  
Loop Structure ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Protein Coding ◽  
Subgenomic Rna ◽  
Stem Loop Structure ◽  
Rna Interaction

ABSTRACT The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.

scholarly journals Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

2008 ◽  
Vol 89 (3) ◽  
pp. 676-686 ◽  
Author(s):  
Kristi Tamm ◽  
Andres Merits ◽  
Inga Sarand
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Structural Protein ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Replicase Complex

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

scholarly journals Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants

2007 ◽  
Vol 81 (6) ◽  
pp. 2849-2860 ◽  
Author(s):  
Giuseppe Balistreri ◽  
Javier Caldentey ◽  
Leevi Kääriäinen ◽  
Tero Ahola
Keyword(s):  
Protease Activity ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Helicase Domain ◽  
Permissive Temperature ◽  
Protease Domain ◽  
Rna Triphosphatase ◽  
Interdomain Interactions

ABSTRACT We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4

Virology ◽  
1990 ◽  
Vol 174 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Dorothea L. Sawicki ◽  
David B. Barkhimer ◽  
Stanley G. Sawicki ◽  
Charles M. Rice ◽  
Sondra Schlesinger
Keyword(s):  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Temperature Sensitive ◽  
Minus Strand

scholarly journals Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1

2008 ◽  
Vol 82 (18) ◽  
pp. 9236-9244 ◽  
Author(s):  
Valeria Lulla ◽  
Dorothea L. Sawicki ◽  
Stanley G. Sawicki ◽  
Aleksei Lulla ◽  
Andres Merits ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Mutant Proteins ◽  
Recombinant Viruses ◽  
Mrna Capping

ABSTRACT Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

scholarly journals RNA Synthesis Directed by a Temperature-Sensitive Mutant of Semliki Forest Virus

1977 ◽  
Vol 37 (2) ◽  
pp. 399-406 ◽  
Author(s):  
J. Saraste ◽  
L. Kaariainen ◽  
H. Soderlund ◽  
S. Keranen
Keyword(s):  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant

scholarly journals Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment

2003 ◽  
Vol 77 (3) ◽  
pp. 1691-1702 ◽  
Author(s):  
Anne Salonen ◽  
Lidia Vasiljeva ◽  
Andres Merits ◽  
Julia Magden ◽  
Eija Jokitalo ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Nonstructural Proteins ◽  
Precursor State ◽  
Replication Complex ◽  
Cytoplasmic Vesicles ◽  
Infected Cells ◽  
Membrane Bound ◽  
Intracellular Vesicles ◽  
The Individual

ABSTRACT The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12CA3 and P12CA34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.

scholarly journals Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons

2009 ◽  
Vol 83 (19) ◽  
pp. 9957-9969 ◽  
Author(s):  
Cara C. Burns ◽  
Ray Campagnoli ◽  
Jing Shaw ◽  
Annelet Vincent ◽  
Jaume Jorba ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Prototype Strain ◽  
Single Step ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Capsid Region ◽  
Replicative Fitness ◽  
Synonymous Codons ◽  
Effective Number Of Codons

ABSTRACT Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

scholarly journals Structural features of NS3 ofDengue virusserotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin

2017 ◽  
Vol 73 (5) ◽  
pp. 402-419 ◽  
Author(s):  
Ankita Pan ◽  
Wuan Geok Saw ◽  
Malathy Sony Subramanian Manimekalai ◽  
Ardina Grüber ◽  
Shin Joon ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Structural Features ◽  
Helicase Domain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Atpase Inhibitors ◽  
The Individual ◽  
Insight Into

Dengue virus(DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains forDengue virusserotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain–domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the174PPAVP179linker stretch of the relatedHepatitis C virus(HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

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