Solubilization and immunoprecipitation of alphavirus replication complexes.

Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsP1, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of approximately 120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsP1, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.     

Expression of the zinc-finger antiviral protein inhibits alphavirus replication

The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.     

Identification and characterization of interferon-induced proteins that inhibit alphavirus replication

Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.     

Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice

Several alphaviruses, including the Sindbis-group viruses, Ross River virus, O'nyong-nyong virus, and Chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. Mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. S.A.AR86 is a Sindbis-group alphavirus closely related to the arthralgia-associated Ockelbo and Girdwood S.A viruses. Following inoculation with S.A.AR86 derived from a molecular clone, infectious virus was isolated from bone and joint tissue 1 to 6 days postinfection. Studies using either in situ hybridization or S.A.AR86-derived double promoter viruses and replicons expressing green fluorescent protein localized sites of viral replication to the periosteum, tendons, and endosteum within the epiphyses of the long bones adjacent to articular joints. These results demonstrate that alphaviruses associated with arthralgia in humans replicate within bone-associated connective tissue adjacent to articular joints in an adult mouse model.     

Salmonid alphavirus replication in mosquito cells: towards a novel vaccine production system

Salmonid alphavirus (SAV) causes pancreas disease and sleeping disease in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) and confers a major burden to the aquaculture industry. A commercial inactivated whole virus vaccine propagated in a salmon cell line at low temperature provides effective protection against SAV infections. Alphaviruses (family Togaviridae) are generally transmitted between vertebrate hosts via blood-sucking arthropod vectors, typically mosquitoes. SAV is unique in this respect because it can be transmitted directly from fish to fish and has no known invertebrate vector. Here, we show for the first time that SAV is able to complete a full infectious cycle within arthropod cells derived from the Asian tiger mosquito Aedes albopictus. Progeny virus is produced in C6/36 and U4.4. cells in a temperature-dependent manner (at 15°C but not at 18°C), can be serially passaged and remains infectious to salmonid Chinook salmon embryo cells. This suggests that SAV is not a vertebrate-restricted alphavirus after all and may have the potential to replicate in invertebrates. The current study also shows the ability of SAV to be propagated in mosquito cells, thereby possibly providing an alternative SAV production system for vaccine applications.     

Ribozyme chemogenetics

In this review I will outline several chemogenetic approaches used to determine the chemical basis of large ribozyme function and structure. The term chemogenetics was first used to describe site-specific functional group modification experiments in the analysis of DNA–protein interactions. Within the past few years equivalent experiments have been performed on large catalytic RNAs using both single-site substitution and interference mapping techniques with nucleotide analogues. While functional group mutagenesis is an important aspect of a chemogenetic approach, chemical correlates to genetic revertants and suppressors must also be realized for the genetic analogy to be intellectually valid and experimentally useful. Several examples of functional group revertants and suppressors have now been obtained within the Tetrahymena group I ribozyme. These experiments define an ensemble of tertiary hydrogen bonds that have made it possible to construct a detailed model of the ribozyme catalytic core. The model includes a functionally important monovalent metal ion binding site, a wobble–wobble receptor motif for helix–helix packing interactions, and a minor groove triple helix. © 1998 John Wiley & Sons, Inc. Biopoly 48: 65–81, 1998     

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Background  

Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.     

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.     

Amiloride inhibition of human cytomegalovirus replication.

Amiloride, an inhibitor of Na+/H+ exchange, interfered with cytomegalovirus (CMV) DNA synthesis, blocked the formation of nuclear inclusions, and reduced CMV infectious yields. The reduction of CMV infectious yields was concentration dependent with an ED90 of 46 microM. Amiloride at a concentration of 150 microM reduced CMV yields by about 100-fold. Reduction of infectious yields appeared to be related to interference with the formation of nuclear inclusions and to inhibition of CMV DNA synthesis. Nuclear inclusions were much reduced in size and demonstrated poorly defined cellulae in the amiloride-treated cells. CMV DNA synthesis was inhibited by approximately 70% when cells were treated with 150 microM amiloride. The reduction in CMV yields could not be related to the reported inhibitory effect of amiloride on protein synthesis. In amiloride (150 microM)-treated, CMV-infected cells, late, yet not immediate-early or early, protein synthesis was markedly decreased relative to untreated, CMV-infected cells. Accordingly, CMV DNA synthesis and the replication of CMV may be related to Na+ entry through an amiloride-sensitive pathway.     

Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer–Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral–neutral–alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.     

Vitamin A inhibition of polyoma virus replication

Vitamin A (retinoic acid) inhibited polyoma virus replication in confluent mouse embryo cells. A significant, dose dependent inhibition was observed when cell monolayers were pretreated with concentrations of vitamin A (10(-8) to 10(-6) M) thought to approximate those found in vivo. This inhibitory effect could be reduced by increasing the input multiplicity of infection. Growth curves of polyoma virus in the presence and absence of vitamin A suggested that vitamin A actually inhibited, and did not simply delay, virus replication. The cell density dependence of this inhibitory effect suggested its association with the prevailing level of cellular DNA synthesis. Vitamin A caused a significant decrease in overall (viral plus cellular) DNA synthesis. Other viruses which do not require induction of host cell DNA synthesis for their replication in confluent, non-dividing cells were not inhibited by vitamin A. These results are consistent with the known inhibitory effects of vitamin A on papovavirus infection in vivo and suggest a mechanism of vitamin A action at the level of the infected cell.     

CNS-Specific Ribozyme Expression

Targeted genetic deletion is a powerful tool for analysis of gene function, but the standard approaches carry certain inescapable disadvantages. First, deletion is ubiquitous; tissue-specific knockout cannot be obtained. Second, temporal regulation of depletion is unattainable; the deleted functions are absent throughout the animal's development. As a consequence, during ontogeny, other gene products may be able to compensate, filling the functional gap. Furthermore bifunctional proteins exist that fulfill one role during development and another in the mature organism; deletion will remove the early function and, if this is lethal, the later function will remain undetected. Third, if genes utilize alternative splicing to control protein expression, it is difficult to target one spliced mRNA while leaving intact its related, but different, siblings. We review how these problems may be circumvented using ribozymes to diminish gene expression in a tissue-specific and temporally regulated manner and provide guidelines for the design and delivery of active ribozymesin vivo.Such methods may be particularly useful for analysis of genes involved in ontogeny and function of the central nervous system, in which individual genes may be expressed with alternative splicing patterns, or at differentially regulated levels, at different stages of CNS development.     

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.     

Ribozyme speed limits

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.     

Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells

For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.