Andres Merits

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein

Plant virus‐encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall‐associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell‐to‐cell movement of tobacco mosaic virus.

Identification of a novel function of the Alphavirus capping apparatus. RNA 5′-triphosphatase activity of Nsp2.

Both genomic and subgenomic RNAs of theAlphavirus have m7G(5′)ppp(5′)N (cap0 structure) at their 5′ end. Previously it has been shown thatAlphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5′ γ-phosphate of the nascent viral RNA chain is removed by a specific RNA 5′-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the γ,β-triphosphate bond at the 5′ end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1–470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471–799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment

ABSTRACT The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12CA3 and P12CA34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

ABSTRACT Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m7GTP or m7GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m7GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m7GMP, in the presence of AdoMet and GTP, because radioactivity from both [α-32P]GTP and [3H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m7GTP, m7GDP, et2m7GMP, and m2et7GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

A Pathogenic Role for CD4+ T Cells during Chikungunya Virus Infection in Mice

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Injury to the joint is believed to occur because of viral and host immune-mediated effects. However, the exact involvement of the different immune mediators in CHIKV-induced pathogenesis is unknown. In this study, we assessed the roles of T cells in primary CHIKV infection, virus replication and dissemination, and virus persistence, as well as in the mediation of disease severity in adult RAG2−/−, CD4−/−, CD8−/−, and wild-type CHIKV C57BL/6J mice and in wild-type mice depleted of CD4+ or CD8+ T cells after Ab treatment. CHIKV-specific T cells in the spleen and footpad were investigated using IFN-γ ELISPOT. Interestingly, our results indicated that CHIKV-specific CD4+, but not CD8+, T cells are essential for the development of joint swelling without any effect on virus replication and dissemination. Infection in IFN-γ−/− mice demonstrated that pathogenic CD4+ T cells do not mediate inflammation via an IFN-γ–mediated pathway. Taken together, these observations strongly indicate that mechanisms of joint pathology induced by CHIKV in mice resemble those in humans and differ from infections caused by other arthritogenic viruses, such as Ross River virus.

Viperin restricts chikungunya virus replication and pathology

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease.

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C(478)A, (P2(CA)) completely abolished the processing of P12(CA)34, P12(CA)3 and P2(CA)3. Co-expression of P23 and P12(CA)34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D(6)A, (P1234(DA)) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection.

Semliki Forest virus nsP3 sequesters G3BP to inhibit stress granule formation on viral mRNAs. Furthermore, the efficient translation of viral mRNAs containing a translation enhancer element assists disruption of SGs in infected cells. This work thus describes a novel mechanism for SG disruption.

Phosphorylation Down-regulates the RNA Binding Function of the Coat Protein of Potato Virus A

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [33P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylatedin vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn2+over Mg2+, can be inhibited by micromolar concentrations of Zn2+ and Cd2+, and is not Ca2+-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.