Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication.

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose-mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human ADP-ribose glycohydrolase MacroD1 inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression. The inhibitor was similarly active in alphavirus trans-replication systems, indicating that it targets the viral RNA replication stage.

Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery.

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs.

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes.

Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and in vitro replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

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.

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments.

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC50] = 0.03 µM) and Semliki Forest virus (SFV; EC50 = 0.11 µM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins.

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling.

Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE: Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections.

Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales.

UNLABELLED: The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCE: Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

Design and Validation of Novel Chikungunya Virus Protease Inhibitors.

Chikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC50) of the most potent inhibitor being ∼1.5 µM. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism.

Ability of minus strands and modified plus strands to act as templates in Semliki Forest virus RNA replication.

During virus multiplication, the viral genome is recognized and recruited for replication based on specific cis-acting elements. Here, we dissected the important cis-acting sequence elements in Semliki Forest virus RNA by using a trans-replication system. As the viral replicase is expressed from a separate plasmid, the template RNA can be freely modified in this system. We show that the cis-acting element at the beginning of the non-structural protein 1 (nsP1) coding region together with the end of the 3' UTR are the minimal requirements for minus-strand synthesis. To achieve a high level of replication, the native 5' UTR was also needed. The virus-induced membranous replication compartments (spherules) were only detected when a replication-competent template was present with an active replicase and minus strands were produced. No translation could be detected from the minus strands, suggesting that they are segregated from the cytoplasm. Minus strands could not be recruited directly to initiate the replication process. Thus, there is only one defined pathway for replication, starting with plus-strand recognition followed by concomitant spherule formation and minus-strand synthesis.

Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization.

UNLABELLED: Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE: SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that both SFV and CHIKV activate the prosurvival PI3K-Akt-mTOR pathway in cells but greatly differ in their capacities to do so: Akt is strongly and persistently activated by SFV infection but only moderately activated by CHIKV. We mapped this activation capacity to a region in nonstructural protein 3 (nsP3) of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalized from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localization may have implications for pathology.

Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis.

UNLABELLED: The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071-8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294) . In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo. IMPORTANCE: Coronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.

Correlative light and electron microscopy enables viral replication studies at the ultrastructural level.

Electron microscopy (EM) is a powerful tool to study structural changes within cells caused e.g. by ectopic protein expression, gene silencing or virus infection. Correlative light and electron microscopy (CLEM) has proven to be useful in cases when it is problematic to identify a particular cell among a majority of unaffected cells at the EM level. In this technique the cells of interest are first identified by fluorescence microscopy and then further processed for EM. CLEM has become crucial when studying positive-strand RNA virus replication, as it takes place in nanoscale replication sites on specific cellular membranes. Here we have employed CLEM for Semliki Forest virus (SFV) replication studies both by transfecting viral replication components to cells or by infecting different cell types. For the transfection-based system, we developed an RNA template that can be detected in the cells even in the absence of replication and thus allows exploration of lethal mutations in viral proteins. In infected mammalian and mosquito cells, we were able to find replication-positive cells by using a fluorescently labeled viral protein even in the cases of low infection efficiency. The fluorescent region within these cells was shown to correspond to an area rich in modified membranes. These results show that CLEM is a valuable technique for studying virus replication and membrane modifications at the ultrastructural level.

The C-terminal repeat domains of nsP3 from the Old World alphaviruses bind directly to G3BP.

The Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding.

Template RNA length determines the size of replication complex spherules for Semliki Forest virus.

The replication complexes of positive-strand RNA viruses are always associated with cellular membranes. The morphology of the replication-associated membranes is altered in different ways in different viral systems, but many viruses induce small membrane invaginations known as spherules as their replication sites. We show here that for Semliki Forest virus (SFV), an alphavirus, the size of the spherules is tightly connected with the length of the replicating RNA template. Cells with different model templates, expressed in trans and copied by the viral replicase, were analyzed with correlative light and electron microscopy. It was demonstrated that the viral-genome-sized template of 11.5 kb induced spherules that were ∼58 nm in diameter, whereas a template of 6 kb yielded ∼39-nm spherules. Different sizes of viral templates were replicated efficiently in trans, as assessed by radioactive labeling and Northern blotting. The replication of two different templates, in cis and trans, yielded two size classes of spherules in the same cell. These results indicate that RNA plays a crucial determining role in spherule assembly for SFV, in direct contrast with results from other positive-strand RNA viruses, in which either the presence of viral RNA or the RNA size do not contribute to spherule formation.

Structure-function analysis of severe acute respiratory syndrome coronavirus RNA cap guanine-N7-methyltransferase.

Coronaviruses possess a cap structure at the 5' ends of viral genomic RNA and subgenomic RNAs, which is generated through consecutive methylations by virally encoded guanine-N7-methyltransferase (N7-MTase) and 2'-O-methyltransferase (2'-O-MTase). The coronaviral N7-MTase is unique for its physical linkage with an exoribonuclease (ExoN) harbored in nonstructural protein 14 (nsp14) of coronaviruses. In this study, the structure-function relationships of the N7-MTase were analyzed by deletion and site-directed mutagenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp14. The results showed that the ExoN domain is closely involved in the activity of the N7-MTase, suggesting that coronavirus N7-MTase is different from all other viral N7-MTases, which are separable from other structural domains located in the same polypeptide. Two of the 12 critical residues identified to be essential for the N7-MTase were located at the N terminus of the core ExoN domain, reinforcing a role of the ExoN domain in the N7-MTase activity of nsp14. The other 10 critical residues were distributed throughout the N7-MTase domain but localized mainly in the S-adenosyl-l-methionine (SAM)-binding pocket and key structural elements of the MTase fold of nsp14. The sequence motif DxGxPxA (amino acids [aa] 331 to 338) was identified as the key part of the SAM-binding site. These results provide insights into the structure and functional mechanisms of coronaviral nsp14 N7-MTase.

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection.

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1.

Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse-chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER-Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.

The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.