Tero Ahola

Viral RNA Replication in Association with Cellular Membranes

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a “tip-anchored” protein attached to the membrane by an amphipathic α-helix (NS5A) and a “tail-anchored” posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic α-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

The Crystal Structures of Chikungunya and Venezuelan Equine Encephalitis Virus nsP3 Macro Domains Define a Conserved Adenosine Binding Pocket

ABSTRACT Macro domains (also called “X domains”) constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an “Old World” alphavirus) and the Venezuelan equine encephalitis virus (a “New World” alphavirus) were determined at resolutions of 1.65 and 2.30 Å, respectively. These domains are active as adenosine di-phosphoribose 1″-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1″-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.

Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes

ABSTRACT Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.

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.

Functions of alphavirus nonstructural proteins in RNA replication

Publisher Summary Alphaviruses are enveloped positive-strand RNA viruses transmitted to vertebrate hosts by mosquitoes. Several alphaviruses are pathogenic to humans or domestic animals, causing serious central nervous system infections or milder infections, for example, arthritis, rash, and fever. The structure and replication of Semliki Forest virus (SFV) and Sindbis virus (SIN) have been studied extensively during the past 30 years. Alphaviruses have been important probes in cell biology to study the translation, glycosylation, folding, and transport of membrane glycoproteins, as well as endocytosis and membrane fusion mechanisms. A new organelle, the intermediate compartment, operating between the endoplasmic retieulum and the Golgi complex has been found by the aid of SFV. During the past 10 years, alphavirus replicons have been increasingly used as expression vectors for basic research, for the generation of vaccines, and for the production of recombinant proteins in industrial scale. The main approaches of laboratories in the recent years have been twofold. On one hand, they have discovered and characterized the enzymatic activities of the individual replicase proteins and on the other hand, they have studied the localization, membrane association, and other cell biological aspects of the replication complex.

Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase

The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3′-to-5′ exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.

Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping

ABSTRACT Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal geneXRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

The Effects of Palmitoylation on Membrane Association of Semliki Forest Virus RNA Capping Enzyme

The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes. To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue. Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys418, Cys419, and Cys420 resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mM sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein. Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.

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.