Replicase Proteins Under Scrutiny : Trans-Replication Systems to Dissect RNA Virus Replication

My doctoral thesis examines the prerequisites of replication for three positive-strand RNA viruses, Chikungunya virus (CHIKV - alphavirus), Semliki Forest virus (SFV - alphavirus) and Flock House virus (FHV - nodavirus). Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes high fever, rashes and joint pain. Semliki Forest virus (SFV) has been extensively studied as a model to comprehend the replication strategies of alphaviruses because of its low pathogenicity. A characteristic feature of alphavirus replication is the formation of membranous invaginations termed spherules, associated with the plasma membrane. Spherules act as genome factories as they are the sites of active viral replication and release nascent viral RNA strands into the cytoplasm through a bottleneck-like structure. We created a trans-replication system specific for CHIKV that would be flexible and presents no danger to the scientist. In this system, the viral replicase proteins are expressed from a DNA plasmid while the RNA template is produced from a second plasmid, in mammalian cells. This allowed for the study of viral replication without generating infectious particles. It also enabled the visualisation of spherules and labelling of all viral replicase proteins with fluorescent or small immunological tags while preserving their function. Various mutations associated with noncytotoxic phenotypes were analysed and the results showed no correlation between the level of RNA replication and cytotoxicity. Moreover, the trans-replication system was used to elucidate that the cysteine residue of CHIKV nsP2 at position 478 is responsible for its protease activity and essential for replicase polyprotein processing. Trp479 of nsP2 also plays a vital role in RNA replication. The insect nodavirus, FHV, verges upon the properties of a ‘universal virus’ as it can replicate in a wide range of hosts. Only the replicase protein A is required for its replication. An efficient FHV trans-replication system was established in mammalian cells. The outer surface of mitochondria displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. High-level synthesis of both genomic and subgenomic RNA was detected in vitro using mitochondrial pellets isolated from transfected cells. The newly synthesized RNA was found to be of positive polarity. This system was used to investigate the capping enzyme domain of protein A, both in cells and in vitro. Mutating the most conserved amino acids of the capping domain abolished or reduced viral RNA synthesis. Surprisingly, transfection of capped RNA template did not rescue the replication activity of the mutants. FHV and alphaviruses show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. The biological systems presented in this study offer valuable knowledge that could be exploited to understand the replication of other RNA viruses and also open up new avenues for the elucidation of key virus-host interactions.Väitöskirjani käsittelee kolmen positiivisäikeisen RNA-viruksen lisääntymistä. Nämä virukset ovat alfaviruksiin kuuluvat chikungunya virus (CHIKV) ja Semliki Forest virus (SFV) sekä nodaviruksiin kuuluva Flock House virus (FHV). CHIKV on hyttyslevitteinen virus, joka aiheuttaa kovaa kuumetta, ihottumaa ja nivelkipuja. SFV:tä on käytetty tutkimuksissa paljon malliviruksena, koska se ei aiheuta tautia ihmisille. Alfavirusten lisääntymisen aikana solukalvolle syntyy sferuleiksi kutsuttuja kalvokuroumia. Ne toimivat viruksen genomia kopioivina tehtaina, joista tuoreet RNA:t vapautuvat solun sytoplasmaan ohuen kaulamaisen rakenteen kautta. Olemme kehittäneet replikaatiosysteemeitä (ns. trans-replikaatio), jotka ovat bioturvallisia ja joustavia tutkimusvälineitä. Näissä systeemeissä viruksen lisääntymisproteiinit ilmennetään DNA-plasmidista, kun taas toinen plasmidi tuottaa kopioituvan RNA-templaatin. Soluissa ei synny infektiokykyisiä viruksia. CHIKV-systeemissä voitiin nähdä runsaasti sferuleita, ja kaikki lisääntymisproteiinit voitiin leimata eri tavoilla. Lisäksi systeemissä analysoitiin mutaatioita, jotka vähensivät viruksen haitallisia vaikutuksia solulle, mutta nämä vaikutukset eivät korreloineet lisääntymistason kanssa. Toisaalta osoitettiin vielä, että viruksen proteaasin nsP2:n aktiivinen kohta on kysteiinitähde 478. Hyönteisvirus FHV lähestyy universaalia virusta, sillä se pystyy lisääntymään monen tyyppisissä isäntäsoluissa. Vain yksi lisääntymisproteiini (nimeltään proteiini A) tarvitaan lisääntymiseen. Rakensimme tehokkaan trans-replikaatiosysteemin FHV:lle nisäkässoluissa. Solun mitokondrioiden ulkokalvolla näkyi runsaasti kalvokuroumia, joiden avonainen kaula osoitti kohti sytoplasmaa. Soluissa ja solulysaateissa havaittiin tehokas RNA:n kopioituminen. Tässä systeemissä tutkittiin proteiini A:n aktiivisuutta RNA:n capping-reaktiossa mutanttien avulla: mutaatiot estivät tai vähensivät RNA-synteesiä. FHV:llä ja alfaviruksilla on evoluution näkökulmasta mielenkiintoisia samankaltaisuuksia. Tässä työssä kehitetyt biologiset systeemit lisäävät tietoa RNA-viruksista ja siitä miten ne toimivat interaktiossa isäntänsä kanssa

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication.The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a 'neck' structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication.Peer reviewe

Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template

Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins. IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.Peer reviewe

Macrodomain binding compound MRS 2578 inhibits alphavirus replication

Peer reviewe

Macrodomain binding compound MRS 2578 inhibits alphavirus replication

Peer reviewe

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.Peer reviewe

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.Peer reviewe

Visualization of spherules in the cells transfected with <i>trans</i>-replicase vectors.

<p>BSR cells were co-transfected with T7-Rluc-Tom + T7-P12^E34 <b>(A, B)</b>, T7-Rluc-Tom + T7-P1234 <b>(C)</b> or T7-Rluc-Tom-Vis + T7-P123^E4^GAA <b>(D)</b> plasmids. Cells were fixed and analyzed using CLEM at 16 h post transfection. Spherules (indicated by arrows) were detected in all cells where replication of template RNA was initiated (identified by Tomato fluorescence). Characteristic EM images are shown in panels A-C. No spherules were detected in cells co-transfected with T7-Rluc-Tom-Vis+T7-P123^E4^GAA.</p

Tagging of ns proteins in the context of CHIKV replicons and genomes.

<p><b>(A)</b> Drawing above: sites used for insertion of EGFP into nsP1, nsP2 and nsP3 of CHIKV replicon are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151616#pone.0151616.g004" target="_blank">Fig 4A</a>. SG—SG promoter (arrow designates the transcription start site), SP6 –bacteriophage SP6 RNA polymerase promoter. Drawing below: CHIKV replicon encoding for nsP4 with SF- or HF-tag (black) attached to its C-terminus. In these constructs the SG promoter was inactivated by synonymous changes (SC). <b>(B)</b> BHK-21 cells were transfected with the indicated <i>in vitro</i> synthesized replicon RNAs. Cells were lysed upon detection of prominent cytopathic effects, and the proteins expressed were analyzed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151616#pone.0151616.g004" target="_blank">Fig 4D</a>. Data from one independent reproducible experiment out of two is shown.<b>(C)</b> Schematic representation of genomes of wild type CHIKV (ICRES-wt) and recombinant viruses encoding for nsP4 with tags at the C-terminus of the protein. Designations are the same as on panel A. <b>(D)</b> BHK-21 cells were transfected with <i>in vitro</i> synthesized ICRES-4^SF, ICRES-4^HF and ICRES-wt RNAs; mock-transfected cells were used as control. Transfected cells were lysed upon detection of prominent cytopathic effects. nsP2 was detected with a corresponding polyclonal antibody and tagged nsP4 was detected with a monoclonal antibody against the FLAG-tag. Data from one independent reproducible experiment out of two is shown.<b>(E)</b> BHK-21 cells were transfected with <i>in vitro</i> synthesized Repl-4^SF, Repl-4^HF and Repl-wt RNAs and analyzed as described above. Data from one independent reproducible experiment out of two is shown.</p

Expression of marker proteins in cell lines transfected with <i>trans</i>-replicase vectors.

<p>Huh7, U2OS, COP-5, BHK-21 and BSR cells were co-transfected with CMV-P1234 + CMV-Fluc-Gluc (marked as CMV); BSR cells were also co-transfected with T7-P1234 + T7-FLuc-Gluc (marked as T7). Control cells were transfected with T7-FLuc-Gluc or CMV-Fluc-Gluc together with corresponding plasmid encoding for inactive replicase. Cells were lysed at 18 h post transfection. Gluc <b>(A)</b> and Fluc <b>(C)</b> activities (RLU—relative light unit), normalized to the total protein content in the lysate, are shown. Each column represents an average of three independent experiments; error bars represent standard deviation. All differences between P1234^GAA (empty columns) and P1234 (grey columns) were highly significant (p<<0.001); in C ** designates p<0.01 and *** designates p<0.001 (one-way ANOVA Sidak’s multiple comparisons test). <b>(B)</b> and <b>(D)</b>. The reporter activities generated by the active replicase were normalized to those measured with the inactive controls.</p