Organization of theFlavivirusRNA 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.

Download Full-text

The HCV Replicase Complex and Viral RNA Synthesis

Hepatitis C Virus I ◽

10.1007/978-4-431-56098-2_8 ◽

2016 ◽

pp. 149-196

Author(s):

Inés Romero-Brey ◽

Volker Lohmann

Keyword(s):

Rna Synthesis ◽

Viral Rna ◽

Replicase Complex

Download Full-text

Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

Journal of Virology ◽

10.1128/.74.10.4579-4589.2000 ◽

2000 ◽

Vol 74 (10) ◽

pp. 4579-4589 ◽

Cited By ~ 1

Author(s):

Minna T. Tuittila ◽

Maria G. Santagati ◽

Matias Röyttä ◽

Jorma A. Määttä ◽

Ari E. Hinkkanen

Keyword(s):

Semliki Forest Virus ◽

Replicase Complex

Download Full-text

Evidence for interaction between the 2a polymerase protein and the 3a movement protein of Cucumber mosaic virus

Journal of General Virology ◽

10.1099/vir.0.81139-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3171-3177 ◽

Cited By ~ 16

Author(s):

Min Sook Hwang ◽

Sang Hyon Kim ◽

Jeong Hyun Lee ◽

Jung Myung Bae ◽

Kyung Hee Paek ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Viral Genome ◽

Movement Protein ◽

Protein A ◽

Yeast Two Hybrid ◽

Rna Molecules ◽

Infected Cells ◽

Replicase Complex ◽

Two Hybrid

The genome of Cucumber mosaic virus consists of three single-stranded RNA molecules, RNAs 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome and have been implicated in movement of the viral RNAs in plants. The 3a movement protein (MP), encoded by RNA 3, is essential for transferring the RNA genomes from infected cells to adjacent cells across the plasmodesmata. Far-Western analysis demonstrated that bacterially expressed 2a polymerase protein directly interacted with the MP. Interaction was confirmed in a yeast two-hybrid assay, and co-immunoprecipitation analysis showed that the MP interacted only with the 2a polymerase protein. A yeast three-hybrid assay showed that the 1a–2a protein interaction relevant for replicase complex formation was not affected by the MP. Although the MP has no affinity for the 1a protein, it interacted indirectly with the 1a protein via the 2a polymerase protein. These results suggest that the replicase complex may be involved in movement through its interaction with the MP.

Download Full-text

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Acta Biochimica Polonica ◽

10.18388/abp.1998_4345 ◽

1998 ◽

Vol 45 (4) ◽

pp. 847-868 ◽

Cited By ~ 16

Author(s):

M Figlerowicz ◽

J J Bujarski

Keyword(s):

Mosaic Virus ◽

Genetic Recombination ◽

Rna Virus ◽

Brome Mosaic Virus ◽

Template Switching ◽

Rna Recombination ◽

Signal Sequences ◽

Experimental Systems ◽

Replicase Complex ◽

Molecular Aspects

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.

Download Full-text

The Contribution of Ribosomal Protein S1 to the Structure and Function of Qβ Replicase

Acta Naturae ◽

10.32607/20758251-2017-9-4-26-30 ◽

2017 ◽

Vol 9 (4) ◽

pp. 26-30

Author(s):

Z. Sh. Kutlubaeva ◽

Е. V. Chetverina ◽

A. B. Chetverin

Keyword(s):

Crystal Structure ◽

Ribosomal Protein ◽

Rna Binding ◽

Structure And Function ◽

Ribosomal Protein S1 ◽

Bacterial Ribosome ◽

New Findings ◽

Replicase Complex ◽

And Function ◽

Fixed Structure

The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Q replicase, the RNA-directed RNA polymerase of bacteriophage Q. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Q replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

Download Full-text

Nonstructural protein 7 and 8 complexes of SARS-CoV-2

10.21203/rs.3.rs-96734/v1 ◽

2020 ◽

Author(s):

Changhui Zhang ◽

Li Li ◽

Jun He ◽

Cheng Chen ◽

Dan Su

Keyword(s):

Nonstructural Protein ◽

Genome Replication ◽

Global Public Health ◽

Host Proteins ◽

Health Crisis ◽

Public Health Crisis ◽

C Terminus ◽

Viral Genome Replication ◽

Replicase Complex ◽

Viral Replicase

Abstract The pandemic outbreak of coronavirus disease 2019 (COVID-19) across the world has led to millions of infection cases and caused a global public health crisis. Current research suggests that SARS-CoV-2 is a highly contagious coronavirus that spreads rapidly through communities. To understand the mechanisms of viral replication, it is imperative to observe coronavirus viral replicase, a huge protein complex comprising up to 16 viral nonstructural and associated host proteins, which is the most promising antiviral target for inhibiting viral genome replication and transcription. Recently, several components of the viral replicase complex in SARS-CoV-2 have been solved to provide a basis for the design of new antiviral therapeutics. Here, we report the crystal structure of the SARS-CoV2 nsp7-8 tetramer, which comprises two copies of each protein representing nsp7’s full-length and the C-terminus of nsp8 owing to N-terminus proteolysis during the process of crystallization. We also identified a long helical extension and highly flexible N-terminal domain of nsp8, which is preferred for interacting with single-stranded nucleic acids.

Download Full-text