Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant

Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

Abstract Background Cellular membranes are crucial host components utilized by positive-strand RNA viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of positive-strand RNA virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with positive-strand RNA virus replication been examined in detail. Results In this study we used parallel and complementary global and targeted approaches to examine the impact of lipid metabolism on the replication of the well-studied model alphanodavirus Flock House virus (FHV). We found that FHV RNA replication in cultured Drosophila S2 cells stimulated the transcriptional upregulation of several lipid metabolism genes, and was also associated with increased phosphatidylcholine accumulation with preferential increases in lipid molecules with longer and unsaturated acyl chains. Furthermore, targeted RNA interference-mediated downregulation of candidate glycerophospholipid metabolism genes revealed a functional role of several genes in virus replication. In particular, we found that downregulation of Cct1 or Cct2, which encode essential enzymes for phosphatidylcholine biosynthesis, suppressed FHV RNA replication. Conclusion These results indicate that glycerophospholipid metabolism, and in particular phosphatidylcholine biosynthesis, plays an important role in FHV RNA replication. Furthermore, they provide a framework in which to further explore the impact of specific steps in lipid metabolism on FHV replication, and potentially identify novel cellular targets for the development of drugs to inhibit positive-strand RNA viruses.http://deepblue.lib.umich.edu/bitstream/2027.42/78268/1/1471-2164-11-183.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78268/2/1471-2164-11-183-S3.XLShttp://deepblue.lib.umich.edu/bitstream/2027.42/78268/3/1471-2164-11-183-S2.XLShttp://deepblue.lib.umich.edu/bitstream/2027.42/78268/4/1471-2164-11-183.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/78268/5/1471-2164-11-183-S4.XLShttp://deepblue.lib.umich.edu/bitstream/2027.42/78268/6/1471-2164-11-183-S1.XLSPeer Reviewe

Truncation of gene F5L partially masks rescue of vaccinia virus strain MVA growth on mammalian cells by restricting plaque size

Modified vaccinia virus Ankara (MVA) is a candidate vaccine vector that is severely attenuated due to mutations acquired during several hundred rounds of serial passage in vitro. A previous study used marker rescue to produce a set of MVA recombinants with improved replication on mammalian cells. Here, we extended the characterization of these rescued MVA strains and identified vaccinia virus (VACV) gene F5L as a determinant of plaque morphology but not replication in vitro. F5 joins a growing group of VACV proteins that influence plaque formation more strongly than virus replication and which are disrupted in MVA. These defective genes in MVA confound the interpretation of marker rescue experiments designed to map mutations responsible for the attenuation of this important VACV strain.This work was funded by grants to DCT: NHMRC APP1023141 and ARC FT110100310

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex

Mutagenesis of the conserved 51-nucleotide region of Sindbis virus

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication

Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses

The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae)

Enhancement of vaccinia virus based oncolysis with histone deacetylase inhibitors

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells. © 2010 MacTavish et al