Virulence variability of the Drosophila C virus and effects of the microparasite on demographic parameters of the host (Drosophila melanogaster)

We carried out experiments with the Drosophila C virus (DCV), a nonhereditary virus acting on demographic parameters of infected Drosophila host populations. It is well known that DCV increases mortality rate, decreases developmental time, and increases daily fecundity. As usual for Drosophila viruses, the DCV was multiplied in vivo. In this study we tested the hypothesis of virulence variability in DCV strains by isolating different stocks of the virus. The flies were tested for susceptibility to injection of such isolates and for virulence variability. Possible interactions between demographic parameters in three Drosophila host populations and injected isolates were studied under two egg densities (low and high). The hypothesis of virulence variability of DCV was supported by significant differences in mortality rates, depending on whether virus isolates were ingested or injected. When DCV was ingested, differences between host mortality rates were independent of the Drosophila host populations. Nevertheless, the developmental time was equally decreased by each virus isolate, independent of the host population. Moreover, the two viral stocks strongly increased the egg production of the flies. This experimental approach clearly showed that DCV could be considered a polymorphic virus. The phenotypic interactions between DCV and host flies varied according to parasite genotype.     

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high‐energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA. However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La‐related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI‐Larp complex promotes the synthesis of a subset of nuclear‐encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI‐Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron‐transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI‐Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis.     

Cricket Paralysis Virus, a Potential Control Agent for the Olive Fruit Fly, Dacus oleae Gmel

Representatives of several families of insect viruses were tested for growth and pathogenicity in the olive fruit fly, Dacus oleae Gmel. The viruses included nuclear polyhedrosis viruses, an iridovirus, two picornaviruses, and Trichoplusia ni small RNA virus (a member of the Nudaurelia β family), in addition to two naturally occurring viruses of the olive fruit fly. Two viruses, one of the two picornaviruses (cricket paralysis virus [CrPV] and the iridovirus (type 21 from Heliothis armigera), were found to replicate in adult flies. Flies which were fed on a solution containing CrPV for 1 day demonstrated a high mortality with 50% dying within 5 days and nearly 80% dying within 12 days of being fed. The virus was transmissible from infected to noninfected flies by fecal contamination. The CrPV which replicated in the infected flies was demonstrated to be the same as input virus by infection of Drosophila melanogaster cells and examination of the expressed viral proteins, immunoprecipitation of the virus purified from flies, and electrophoretic analysis of the structural proteins.     

Sex-specific behavioural symptoms of viral gut infection and Wolbachia in Drosophila melanogaster

All organisms are infected with a range of symbionts spanning the spectrum of beneficial mutualists to detrimental parasites. The fruit fly Drosophila melanogaster is a good example, as both endosymbiotic Wolbachia, and pathogenic Drosophila C Virus (DCV) commonly infect it. While the pathophysiology and immune responses against both symbionts are the focus of intense study, the behavioural effects of these infections have received less attention. Here we report sex-specific behavioural responses to these infections in D. melanogaster. DCV infection caused increased sleep in female flies, but had no detectable effect in male flies. The presence of Wolbachia did not reduce this behavioural response to viral infection. We also found evidence for a sex-specific cost of Wolbachia, as male flies infected with the endosymbiont became more lethargic when awake. We discuss these behavioural symptoms as potentially adaptive sickness behaviours.     

Symbionts Commonly Provide Broad Spectrum Resistance to Viruses in Insects: A Comparative Analysis of Wolbachia Strains

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses – DCV and FHV – was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection — activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia''s antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.     

Variation in Antiviral Protection Mediated by Different Wolbachia Strains in Drosophila simulans

Drosophila C virus (DCV) is a natural pathogen of Drosophila and a useful model for studying antiviral defences. The Drosophila host is also commonly infected with the widespread endosymbiotic bacteria Wolbachia pipientis. When DCV coinfects Wolbachia-infected D. melanogaster, virus particles accumulate more slowly and virus induced mortality is substantially delayed. Considering that Wolbachia is estimated to infect up to two-thirds of all insect species, the observed protective effects of Wolbachia may extend to a range of both beneficial and pest insects, including insects that vector important viral diseases of humans, animals and plants. Currently, Wolbachia-mediated antiviral protection has only been described from a limited number of very closely related strains that infect D. melanogaster. We used D. simulans and its naturally occurring Wolbachia infections to test the generality of the Wolbachia-mediated antiviral protection. We generated paired D. simulans lines either uninfected or infected with five different Wolbachia strains. Each paired fly line was challenged with DCV and Flock House virus. Significant antiviral protection was seen for some but not all of the Wolbachia strain-fly line combinations tested. In some cases, protection from virus-induced mortality was associated with a delay in virus accumulation, but some Wolbachia-infected flies were tolerant to high titres of DCV. The Wolbachia strains that did protect occurred at comparatively high density within the flies and were most closely related to the D. melanogaster Wolbachia strain wMel. These results indicate that Wolbachia-mediated antiviral protection is not ubiquitous, a finding that is important for understanding the distribution of Wolbachia and virus in natural insect populations.     

Dietary Cholesterol Modulates Pathogen Blocking by Wolbachia

The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This “pathogen blocking” could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2–5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.     

A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.     

The heat shock protein 70 cochaperone YDJ1 is required for efficient membrane-specific flock house virus RNA replication complex assembly and function in Saccharomyces cerevisiae

The assembly of RNA replication complexes on intracellular membranes is an essential step in the life cycle of positive-sense RNA viruses. We have previously shown that Hsp90 chaperone complex activity is essential for efficient Flock House virus (FHV) RNA replication in Drosophila melanogaster S2 cells. To further explore the role of cellular chaperones in viral RNA replication, we used both pharmacologic and genetic approaches to examine the role of the Hsp90 and Hsp70 chaperone systems in FHV RNA replication complex assembly and function in Saccharomyces cerevisiae. In contrast to results with insect cells, yeast deficient in Hsp90 chaperone complex activity showed no significant decrease in FHV RNA replication. However, yeast with a deletion of the Hsp70 cochaperone YDJ1 showed a dramatic reduction in FHV RNA replication that was due in part to reduced viral RNA polymerase accumulation. Furthermore, the absence of YDJ1 did not reduce FHV RNA replication when the viral RNA polymerase and replication complexes were retargeted from the mitochondria to the endoplasmic reticulum. These results identify YDJ1 as an essential membrane-specific host factor for FHV RNA replication complex assembly and function in S. cerevisiae and are consistent with known differences in the role of distinct chaperone complexes in organelle-specific protein targeting between yeast and higher eukaryotes.     

Cricket paralysis virus and drosophila C virus: serological analysis and comparison of capsid polypeptides and host range.

Five strains of Drosophila C virus (DCV) were found to be serologically indistinguishable. By using antisera against DVC strains and cricket paralysis virus (CrPV), a relationship was shown to exist between the two viruses. All DCV strains exhibited the same polypeptide profile when analyzed on SDS-polyacrylamide gels and, while basically similar, analysis of CrPV polypeptides revealed that they were slightly larger than those of DCV. Three strains of DCV could be distinguished from each other by their pathogenicity in Drosophila melanogaster or Galleria mellonella, while only CrPV was able to multiply in Gryllus bimaculatus. Also, CrPV but not DCV, could multiply in an established cell line of Lymantria dispar.     

Interaction between a picornavirus and a wild population ofDrosophila melanogaster

Summary Drosophila C virus (DCV) has a considerable impact on ovarian morphogenesis inDrosophila melanogaster host populations. This virus also affects the developmental time and the fresh weight of infected females. In order to investigate the hypothesis that DCV may play a role in the dynamics ofDrosophila populations, the fertility and embryonic and larvo-pupal death rates of a host population and that of five DCV-free populations were determined. A comparison of two populations, one of them DCV-free, the other infected, suggested that the fertility of the DCV-infected flies was higher than that of uninfected flies, despite a greater larvo-pupal death rate. Fertility of the infected flies was greater among the infected population than for the DCV-free populations. The DCV-free populations originated from five different localities. The virus clearly does have an impact on the biotic potential of its host population. This paper reports for the first time a positive interaction between a viral population and a host population as it increases certain parameters of host population dynamics.     

Innovative Toolbox for the Quantification of Drosophila C Virus,Drosophila A Virus,and Nora Virus

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.     

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus.The dependence of viruses on the host translation system imposes constraints that are central to virus biology and have led to specialized mechanisms and intricate regulatory interactions. Failure to translate viral mRNAs and to modulate host mRNA translation would have catastrophic effects on virus replication, spread, and evolution. Accordingly, a wide assortment of virus-encoded functions is dedicated to commandeering and controlling the cellular translation apparatus. Viral strategies to dominate the host translation machinery target the initiation, elongation, and termination steps and include mechanisms ranging from the manipulation of key eukaryotic translation factors to the evolution of specialized cis-acting elements that recruit ribosomes or modify genome-coding capacity. Because many of these strategies have likely been pirated from their hosts and because virus genetic systems can be manipulated with relative ease, the study of viruses has been a preeminent source of information on the mechanism and regulation of the protein synthesis machinery. In this article, we focus on select viruses that infect mammalian or plant cells and review the mechanisms they use to exploit and control the cellular protein synthesis machinery.     

Cyclophilin A Binds to the Viral RNA and Replication Proteins,Resulting in Inhibition of Tombusviral Replicase Assembly

Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.     

Two viral kinases are required for sustained long distance axon transport of a neuroinvasive herpesvirus

Axonal transport is essential for the successful establishment of neuroinvasive herpesvirus infections in peripheral ganglia (retrograde transport) and the subsequent spread to exposed body surfaces following reactivation from latency (anterograde transport). We examined two components of pseudorabies virus (US3 and UL13), both of which are protein kinases, as potential regulators of axon transport. Following replication of mutant viruses lacking kinase activity, newly assembled capsids displayed an increase in retrograde motion that prevented efficient delivery of capsids to the distal axon. The aberrant increase in retrograde motion was accompanied by loss of a viral membrane marker from the transported capsids, indicating that the viral kinases allow for efficient anterograde transport by stabilizing membrane–capsid interactions during the long transit from the neuron cell body to the distal axon.     

A Novel System for the Launch of Alphavirus RNA Synthesis Reveals a Role for the Imd Pathway in Arthropod Antiviral Response

Alphaviruses are RNA viruses transmitted between vertebrate hosts by arthropod vectors, primarily mosquitoes. How arthropods counteract alphaviruses or viruses per se is not very well understood. Drosophila melanogaster is a powerful model system for studying innate immunity against bacterial and fungal infections. In this study we report the use of a novel system to analyze replication of Sindbis virus (type species of the alphavirus genus) RNA following expression of a Sindbis virus replicon RNA from the fly genome. We demonstrate deficits in the immune deficiency (Imd) pathway enhance viral replication while mutations in the Toll pathway fail to affect replication. Similar results were observed with intrathoracic injections of whole virus and confirmed in cultured mosquito cells. These findings show that the Imd pathway mediates an antiviral response to Sindbis virus replication. To our knowledge, this is the first demonstration of an antiviral role for the Imd pathway in insects.     

Viral subversion of the host protein synthesis machinery

Viruses are fully reliant on the translation machinery of their host cells to produce the polypeptides that are essential for viral replication. Consequently, viruses recruit host ribosomes to translate viral mRNAs, typically using virally encoded functions to seize control of cellular translation factors and the host signalling pathways that regulate their activity. This not only ensures that viral proteins will be produced, but also stifles innate host defences that are aimed at inhibiting the capacity of infected cells for protein synthesis. Remarkably, nearly every step of the translation process can be targeted by virally encoded functions. This Review discusses the diverse strategies that viruses use to subvert host protein synthesis functions and regulate mRNA translation in infected cells.