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International audience; Background and Aims: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and known as the viral replicase. The RNA-dependent RNA polymerase (RdRp) domain is responsible for the synthesis of viral RNA.Approach and Results: Here, we identified a highly conserved α-helix located in the RdRp thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RdRp palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release.Conclusions: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RdRp with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RdRp thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to 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.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses infect members from all three domains of life (Archaea, Prokarya, and Eukarya). The replicase (Rep) from these viruses is responsible for initiating rolling circle replication (RCR) of their genomes. Rep is a multifunctional enzyme responsible for nicking and ligating ssDNA and unwinding double-stranded DNA (dsDNA). We report the structure of porcine circovirus 2 (PCV2) Rep bound to ADP and single-stranded DNA (ssDNA), and Rep bound to ADP and double-stranded DNA (dsDNA). The structures demonstrate Rep to be a member of the superfamily 3 (SF3) of ATPases Associated with diverse cellular Activities (AAA+) superfamily clade 4. At the Rep N terminus is an endonuclease domain (ED) that is responsible for ssDNA nicking and ligation, in the center of Rep is an oligomerization domain (OD) responsible for hexamerization, and at the C terminus is an ATPase domain (AD) responsible for ssDNA/dsDNA interaction and translocation. The Rep AD binds to DNA such that the ED faces the replication fork. The six AD spiral around the DNA to interact with the backbone phosphates from four consecutive nucleotides. Three of the six AD are able to sense the backbone phosphates from the second strand of dsDNA. Heterogeneous classification of the data demonstrates the ED and AD to be mobile. Furthermore, we demonstrate that Rep exhibits basal nucleoside triphosphatase (NTPase) activity. IMPORTANCE CRESS-DNA viruses encompass a significant portion of the biosphere’s virome. However, little is known about the structure of Rep responsible for initiating the RCR of CRESS-DNA viruses. We use cryo-electron microscopy (cryo-EM) to determine the structure of PCV2 Rep in complex with ADP and ss/dsDNA. Our structures demonstrate CRESS-DNA Reps to be SF3 members (clade 4) of the AAA+ superfamily. The structures further provide the mechanism by which CRESS-DNA virus Reps recognize DNA and translocate DNA for genome replication. Our structures also demonstrate the ED and AD of PCV2 Rep to be highly mobile. We propose the mobile nature of these domains to be necessary for proper functioning of Reps. We further demonstrate that Reps exhibit basal NTPase activity. Our studies also provide initial insight into the mechanism of RCR.

Centro de Biotecnología y Genómica de Plantas (CBGP), Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target., This research was funded by MINISTERIO DE CIENCIA E INNOVACIÓN, grant numbers SAF2016-80451-P and -PID2019106068GB-I00 to A.M. and A.A.E.; PID2019-105117RR-C21/AEI/ 10.13039/501100011033 to M.Á.M.-A.; P2018/BAA-4370 and E-RTA-2017-00003-C03 to J.-C.S. UNI VERSIDAD DE CASTILLA-LA MANCHA grant numbers GI20163501, GI20174100, and 2019-GRIN 27080 to A.M. A.A. is supported by a Beatriz Galindo Senior Fellowship (BEAGAL18/00074) from MINISTERIO DE UNIVERSIDADES (Spain, 17 Págs. This article belongs to the Special Issue Viral Replication Complexes

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (&gt, 10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target.

The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development., Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless—as happened occasionally—reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity. IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus., Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyltransferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we cloned and expressed the entire NS5 and the RdRpD in a heterologous system and used purified preparations for protein characterizations. We determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA and that the RdRp activity is not inhibited by sofosbuvir. To further examine the characteristics of USUV polymerase in a more specifically biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed their subcellular location. NS5 is predominantly found in the cytoplasm; a significant proportion is directed to the nucleus, and this translocation involves nuclear location signals (NLS) located at least between the MTase and RdRpD domains.

Plasma membrane-associated cation-binding protein 1-like protein of Nicotiana benthamiana plays a role in defense against Bamboo mosaic virus by binding the viral replicase and restricting viral intercellular movement., To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement.

Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle., Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex. IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

Replication of hepatitis C virus (HCV) is tightly linked to membrane alterations designated the membranous web, harboring the viral replicase complex. In this chapter we describe the morphology and 3D architecture of the HCV-induced replication organelles, mainly consisting of double membrane vesicles, which are generated by a concerted action of the nonstructural proteins NS3 to NS5B. Recent studies have furthermore identified a number of host cell proteins and lipids contributing to the biogenesis of the membranous web, which are discussed in this chapter. Viral RNA synthesis is tightly associated with these membrane alterations and mainly driven by the viral RNA dependent RNA polymerase NS5B. We summarize our current knowledge of the structure and function of NS5B, the role of cis-acting replication elements at the termini of the genome in regulating RNA synthesis and the contribution of additional viral and host factors to viral RNA synthesis, which is still ill defined.

Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades.

The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) in Wuhan, China, which rapidly grew into a global pandemic, marked the third introduction of a virulent coronavirus into the human society, affecting not only the healthcare system, but also the global economy. Although our understanding of coronaviruses has undergone a huge leap after two precedents, the effective approaches to treatment and epidemiological control are still lacking. In this article, we present a succinct overview of the epidemiology, clinical features, and molecular characteristics of SARS-CoV-2. We summarize the current epidemiological and clinical data from the initial Wuhan studies, and emphasize several features of SARS-CoV-2, which differentiate it from SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), such as high variability of disease presentation. We systematize the current clinical trials that have been rapidly initiated after the outbreak of COVID-19 pandemic. Whereas the trials on SARS-CoV-2 genome-based specific vaccines and therapeutic antibodies are currently being tested, this solution is more long-term, as they require thorough testing of their safety. On the other hand, the repurposing of the existing therapeutic agents previously designed for other virus infections and pathologies happens to be the only practical approach as a rapid response measure to the emergent pandemic, as most of these agents have already been tested for their safety. These agents can be divided into two broad categories, those that can directly target the virus replication cycle, and those based on immunotherapy approaches either aimed to boost innate antiviral immune responses or alleviate damage induced by dysregulated inflammatory responses. The initial clinical studies revealed the promising therapeutic potential of several of such drugs, including favipiravir, a broad-spectrum antiviral drug that interferes with the viral replication, and hydroxychloroquine, the repurposed antimalarial drug that interferes with the virus endosomal entry pathway. We speculate that the current pandemic emergency will be a trigger for more systematic drug repurposing design approaches based on big data analysis.

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions.

Many viruses replicate and assemble in subcellular microenvironments called virus factories or ‘viroplasm.’ Virus factories increase the efficiency of replication and at the same time protect viruses from antiviral defenses. We describe how viruses reorganize cellular membrane compartments and cytoskeleton to generate these ‘mini-organelles’ and how these rearrangements parallel cellular responses to stress such as protein aggregation and DNA damage.

• Background To establish successful infection, plant viruses produce profound alterations of host physiology, disturbing unrelated endogenous processes and contributing to the development of disease. In tobamoviruses, emerging evidence suggests that viral-encoded proteins display a great variety of functions beyond the canonical roles required for virus structure and replication. Among these, their modulation of host immunity appears to be relevant in infection progression. • Scope In this review, some recently described effects on host plant physiology of Tobacco mosaic virus (TMV)-encoded proteins, namely replicase, movement protein (MP) and coat protein (CP), are summarized. The discussion is focused on the effects of each viral component on the modulation of host defense responses, through mechanisms involving hormonal imbalance, innate immunity modulation and antiviral RNA silencing. These effects are described taking into consideration the differential spatial distribution and temporality of viral proteins during the dynamic process of replication and spread of the virus. • Conclusion In discussion of these mechanisms, it is shown that both individual and combined effects of viral-encoded proteins contribute to the development of the pathogenesis process, with the host plant's ability to control infection to some extent potentially advantageous to the invading virus. Fil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rodriguez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Venturuzzi, Andrea Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Asurmendi, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

To allow the biochemical and structural characterization of the chymotrypsin-like "main proteinase" (non-structural protein 4; nsp4) of the arterivirus prototype Equine Arteritis Virus (EAV), we developed protocols for the large-scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase was expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag. Following purification, the nsp4 moiety of MBP-nsp4 was successfully used for structural studies [Barrette-Ng, I.H., Ng, K.K.S., Mark, B.L., van Aken, D., Cherney, M.M., Garen, C, Kolodenko, Y., Gorbalenya, A.E., Snijder, E.J., James, M.N.G, 2002. Structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole. J. Biol. Chem. 277, 39960-39966]. Furthermore, both forms of the EAV proteinase were shown to be proteolytically active in two different trans-cleavage assays. Recombinant nsp4 cleaved the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates. In a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the P9-P7' residues of six nsp4 cleavage sites was investigated. The peptide representing the EAV nsp7/8 junction was used to optimize the reaction conditions (pH 7.5, 25mM NaCl, 30% glycerol at 30 degrees C), which resulted in a maximum turnover of 15% of this substrate in 4h, using a substrate to enzyme molar ratio of 24:1. The assays described in this study can be used for a more extensive biochemical characterization of the EAV main proteinase, including studies aiming to identify inhibitors of proteolytic activity.

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

Members of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently known. This replicase is expressed from the viral genome by translation of two large 5__-proximal ORFs into two polyproteins, which are processed by virus-encoded proteases in 13-16 individual nonstructural proteins (nsps). The nsps direct the formation of an RNA-synthesizing complex that mediates viral genome replication, as well as the synthesis of a 3__-coterminal nested set of subgenomic (sg) mRNAs, from which the viral genes located downstream of the replicase gene are expressed. Arteriviruses and coronaviruses presumably employ a unique mechanism of discontinuous minus-strand extension to generate subgenome-length templates for sg mRNA synthesis. This thesis focused on the functional characterization of two replicase subunits and their roles in coupling different processes in the replicative cycle of equine arteritis virus (EAV), the arterivirus prototype. The biological importance of a conserved domain unique to nidoviruses (NendoU), mapping to arterivirus nsp11 and proposed to function as an endoribonuclease, was addressed. We demonstrated the recombinant nsp11 exhibits pyrimidine-specific endoribonuclease activity, and showed the critical importance of the NendoU domain for EAV RNA synthesis. In addition, we identified a multidomain replicase subunit, EAV nsp1, as a key coordinator of EAV genome replication, sg mRNA synthesis, and virus production. Our results reveal that the relative abundance of EAV mRNAs is tightly controlled by nsp1 and is critical for efficient production of new virus particles. The protein was implicated in modulating the accumulation of full-length and subgenome-length minus-strand templates for EAV mRNA synthesis. A protocol for purification of soluble recombinant nsp1, which can be used in future research on the molecular mechanisms of nsp1 function, is described.

Equine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the large replicase polyproteins pp1a and pp1ab. Processing of pp1a and pp1ab is mediated by three viral proteases of which a predicted chymotrypsin-like protease residing in nsp4 releases most non-structural proteins from the replicase polyproteins. To obtain more insight in the biochemical properties of this protease, and viral chymotrypsin-like proteases in general, the three-dimensional structure of nsp4 was determined by X-ray crystallography. The nsp4 three-dimensional structure revealed that the enzyme adopts a chymotrypsin-like fold and possesses an additional C-terminal domain (CTD) not found in most other chymotrypsin-like proteases. The structure revealed also that a connecting stretch of amino acids might facilitate movement of the CTD relative to the rest of the molecule. A site-directed mutagenesis study showed that the nsp4 CTD played a crucial role in EAV replicase processing, but that it was not required for proteloytic activity of the protease per se. Furthermore, the formation of an ion pair between Asp-119 and either Arg-4 in the N-terminus or Arg-203 in the C-terminus was suggested, which could play a role in alternate nsp4 conformations needed for e.g. different cis and trans cleavage activities. Mutations targeting the residues involved in these interactions affected the proteolytic activity of nsp4, but the data were inconclusive regarding the importance of ion pair formation. Processing of the C-terminal half of pp1a (the nsp3-8 region) by nsp4 can proceed following either of two alternative proteolytic pathways. To address the importance of both pathways, various cleavage site mutations were engineered, which were expected to block cleavage by nsp4. The experiments showed that all mutations that blocked processing of the corresponding site in the nsp3-8 precursor also blocked or severely inhibited EAV RNA synthesis. Moreover, evidence was obtained for the presence of a novel, internal nsp4 cleavage site in nsp7, which appears to be conserved among arteriviruses. The theoretical chapters in this thesis introduce the reader to (nido)viruses and nidovirus replicase maturation in particular.

Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3′ end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.

Coutard, B. et al., Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules. © 2007 Elsevier B.V. All rights reserved., This work was supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework Programme (FP6)

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.

Affinity labelling with aldehyde-containing analogs of initiation substrates of nuclear fraction of tick-borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single-hit hydroxylamine hydrolysis, using limited and long-term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the ‘B’-site of NTP-binding motif of viral RNA replicase.