The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.
The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3′ end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3′ CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.
The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG (8-10) VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. The Alphavirus genus of the Togaviridae contains almost 30 species of viruses. These viruses are globally distributed and cause a variety of disease, ranging from mild febrile illness to encephalitis and death. Alphaviruses are arthropod borne and enzootically maintained in a cycle between mosquitoes and birds or small mammals. In vertebrate hosts, infection is acute, producing high viral yields prior to clearance or death. In cultured vertebrate, cells with large amounts of viral RNA are rapidly generated prior to apoptotic cell death. Factors likely contributing to the efficiency of RNA synthesis include the sequestration of machinery in cytoplasmic membrane-associated centers (8,12,13,19) and specific regulation of the viral RNA-dependent RNA polymerase (RdRp) activity. The morphogenesis of viral RNA synthetic complexes and regulation of RNA synthetic activity are integrally linked to programmed proteolytic processing of the viral components of the complex over the course of virus infection.Sindbis virus (SIN) is the type species of the Alphavirus genus and serves as a model for the study of viral RNA synthesis. The SIN genome is 11.7 kb of message-sense RNA that is capped and polyadenylated (49, 51), allowing for efficient translation. Following viral entry and release of the genome to the cytoplasm, the large 5Ј open reading frame of the genomic RNA is translated into the nonstructural polyproteins P123 or P1234, required for viral RNA synthesis. P1234 results from a readthrough event at the 3Ј end of the nsP3-coding region (22,31,50). Approximately 10% of translating ribosomes read through the stop codon to give rise to P1234. Cleavage of the nonstructural polyproteins is catalyzed by the nsP2-associated proteinase activity (3, 16), ultimately resulting in four mature proteins, nsP1, nsP2, nsP3, and nsP4. Cleavage of the polyproteins is sequential and temporally regulated, creating viral RNA synthetic complexes with distinct functions over the course of the single-cell replication cycle (21,23,25,46). Initial cleavage of P1234 releases mature nsP4 and occurs rapidly (2, 45). nsP4 contains the RdRp catalytic core but requires the other nsP proteins for activity. P123 and nsP4 form t...
BackgroundThe essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication.MethodsInfluenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference.ResultsExogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell.ConclusionsThese results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.
We have previously shown that hypoxia and N-methyl-D-aspartate (NMDA) receptor activation induce breakdown of choline-containing phospholipids in rat hippocampus, a process which is mediated by calcium influx and phospholipase A (2) activation. Bilobalide, a constituent of Ginkgo biloba, inhibited this process in a potent manner (Weichel et al., Naunyn-Schmiedeberg's Arch. Pharmacol. 360, 609-615, 1999). In this study, we used fluorescence microscopy and radioactive flux measurements to show that bilobalide does not interfere with NMDA-induced calcium influx. Instead, bilobalide seems to inhibit NMDA-induced fluxes of chloride ions through ligand-operated chloride channels. In our experiments, substitution of chloride in the superfusion medium fully blocked the effect of NMDA on choline release from hippocampal slices, while the presence of chloride transport inhibitors (furosemide, DIDS) was partially antagonistic. The inhibitory effect of bilobalide and of HA-966, a glycine B receptor antagonist, on NMDA-induced choline release was attenuated in the presence of glycine. The inhibitory effect of bilobalide, but not that of HA-966, was also antagonized by GABA. The inhibitory effect of MK-801, an NMDA channel blocker, on choline release was insensitive to glycine. We conclude from our findings that bilobalide inhibits an NMDA-induced chloride flux through glycine/GABA-operated channels, thereby preventing NMDA-induced breakdown of membrane phospholipids. This effect is expected to contribute to the neuroprotective effects of ginkgo biloba extracts.
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