Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Lemm, Julie A.; Bergqvist, Anders; Read, Carol M.; Rice, Charles M.

doi:10.1128/jvi.72.8.6546-6553.1998

Cited by 86 publications

(40 citation statements)

References 40 publications

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“…Previously, a trans-replication system for SIN has been developed by using recombinant vaccinia virus-based expression of replicase proteins and model templates (20,23). This system has yielded much invaluable information on alphavirus RNA replication in intact cells and cell extracts (19,22,24), but the construction and use of recombinant vaccinia viruses complicate its use. No published information is available on the relative efficiency of the RNA synthesis in the vaccinia virus-based system.…”

Section: Discussionmentioning

confidence: 99%

Assembly of Alphavirus Replication Complexes from RNA and Protein Components in a Novel trans -Replication System in Mammalian Cells

Spuul¹,

Balistreri

Hellström³

et al. 2011

J Virol

120

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For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.

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Section: Discussionmentioning

confidence: 99%

Assembly of Alphavirus Replication Complexes from RNA and Protein Components in a Novel trans -Replication System in Mammalian Cells

Spuul¹,

Balistreri

Hellström³

et al. 2011

J Virol

120

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“…The organization of the VEE virus positivesense, single-stranded RNA genome is 5%-methylated cap-nontranslated region (5%NTR)-nonstructural protein 1 (nsP1)-nsP2-nsP3-nsP4-26S junction region-capsid-E3-E2-6K-E1-3%NTR-polyadenylated tail. Analogous to other alphaviruses, nonstructural replicase polyproteins are likely translated directly off the plus strand after viral uncoating, orchestrating complementary minusstrand synthesis and (following additional intracellular processing) subgenomic and genomic plus-strand RNA synthesis (de Groot et al, 1990;Strauss and Strauss, 1994;Lemm et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex

et al. 1999

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The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.

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“…However, it is not known whether multiple polyproteins are required to build up an individual replication complex. A large body of evidence indicates that the polyprotein expression and cleavage strategy regulates viral RNA replication (20,24,25,37,46). The fulllength nonstructural (ns) polyprotein, designated P1234, is translated directly from the viral genomic RNA and becomes cleaved immediately after or during its synthesis.…”

mentioning

confidence: 99%

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

Lulla

Tints

et al. 2006

J Virol

100

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The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1 had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site. Semliki Forest virus (SFV)is an enveloped positive-strand RNA virus of the genus Alphavirus, family Togaviridae. Alphavirus replication relies on the production of replicase proteins in the form of polyprotein precursor(s), which are then coand posttranslationally processed (40). This finally leads to the generation of individual replicase protein subunits and ensures the formation of proper interactions between the cleavage products of a single polyprotein (35). However, it is not known whether multiple polyproteins are required to build up an individual replication complex. A large body of evidence indicates that the polyprotein expression and cleavage strategy regulates viral RNA replication (20,24,25,37,46). The fulllength nonstructural (ns) polyprotein, designated P1234, is translated directly from the viral genomic RNA and becomes cleaved immediately after or during its synthesis. Its primary cleavage products, polyprotein P123 and nsP4, form the early replication complexes synthesizing negative-strand RNA. P123 is first cleaved to yield nsP1 and P23, giving an intermediate complex, nsP1-P23-nsP4, which is capable of both negativestrand and positive-strand synthesis (20,24,46) but which appears to be very short lived in infected cells. P23 is rapidly cleaved into nsP2 and nsP3, and the replication complex is rearranged into a stable form making exclusively positive-sense RNA with the negative strand as a template.

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Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Cited by 86 publications

References 40 publications

Assembly of Alphavirus Replication Complexes from RNA and Protein Components in a Novel trans -Replication System in Mammalian Cells

Assembly of Alphavirus Replication Complexes from RNA and Protein Components in a Novel trans -Replication System in Mammalian Cells

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

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