Insect RNA Viruses Research Articles

Transcriptome profiling reveals infection strategy of an insect maculavirus.

Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.

Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA.

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

Wolbachia elevates host methyltransferase expression to block an RNA virus early during infection.

Wolbachia pipientis is an intracellular endosymbiont known to confer host resistance against RNA viruses in insects. However, the causal mechanism underlying this antiviral defense remains poorly understood. To this end, we have established a robust arthropod model system to study the tripartite interaction involving Sindbis virus and Wolbachia strain wMel within its native host, Drosophila melanogaster. By leveraging the power of Drosophila genetics and a parallel, highly tractable D. melanogaster derived JW18 cell culture system, we determined that in addition to reducing infectious virus production, Wolbachia negatively influences Sindbis virus particle infectivity. This is further accompanied by reductions in viral transcript and protein levels. Interestingly, unchanged ratio of proteins to viral RNA copies suggest that Wolbachia likely does not influence the translational efficiency of viral transcripts. Additionally, expression analyses of candidate host genes revealed D. melanogaster methyltransferase gene Mt2 as an induced host factor in the presence of Wolbachia. Further characterization of viral resistance in Wolbachia–infected flies lacking functional Mt2 revealed partial recovery of virus titer relative to wild-type, accompanied by complete restoration of viral RNA and protein levels, suggesting that Mt2 acts at the stage of viral genome replication. Finally, knockdown of Mt2 in Wolbachia uninfected JW18 cells resulted in increased virus infectivity, thus demonstrating its previously unknown role as an antiviral factor against Sindbis virus. In conclusion, our findings provide evidence supporting the role of Wolbachia–modulated host factors towards RNA virus resistance in arthropods, alongside establishing Mt2’s novel antiviral function against Sindbis virus in D. melanogaster.

A novel picorna-like virus, Riptortus pedestris virus-1 (RiPV-1), found in the bean bug, R. pedestris, after fungal infection

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. This is a novel insect positive-sense single-stranded RNA virus, which we named Riptortus pedestris virus-1 (RiPV-1) (GenBank accession no. KU958718). The genome of RiPV-1 consists of 10,554 nucleotides (nt), excluding the poly(A) tail, which contains a single large open reading frame (ORF) of 10,371 nt encoding a polyprotein (3456 aa) and flanked by 71 and 112 nt at the 5′ and 3′ untranslated regions (UTR), respectively. RiPV-1 genome organization from the 5′ end contains a consensus organization of picorna-like RNA virus helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp), in addition to two putative structural proteins located at the 3′ region and a poly(A) tail at the 3′ end. The viral particles were approximately 30nm in diameter with some dispersal distinctive surface projections. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. RiPV-1 could be found in all stages of lab-reared bean bugs and was detected abundantly in the thorax, abdomen, midgut and fat body, but not in the reproductive organs and muscle. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2–6days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

Moku virus; a new Iflavirus found in wasps, honey bees and Varroa.

There is an increasing global trend of emerging infectious diseases (EIDs) affecting a wide range of species, including honey bees. The global epidemic of the single stranded RNA Deformed wing virus (DWV), driven by the spread of Varroa destructor has been well documented. However, DWV is just one of many insect RNA viruses which infect a wide range of hosts. Here we report the full genome sequence of a novel Iflavirus named Moku virus (MV), discovered in the social wasp Vespula pensylvanica collected in Hawaii. The novel genome is 10,056 nucleotides long and encodes a polyprotein of 3050 amino acids. Phylogenetic analysis showed that MV is most closely related to Slow bee paralysis virus (SBPV), which is highly virulent in honey bees but rarely detected. Worryingly, MV sequences were also detected in honey bees and Varroa from the same location, suggesting that MV can also infect other hymenopteran and Acari hosts.

Binding and entry of a non-enveloped T=4 insect RNA virus is triggered by alkaline pH

Tetraviruses are small, non-enveloped, RNA viruses that exclusively infect lepidopteran insects. Their particles comprise 240 copies of a single capsid protein precursor (CP), which undergoes autoproteolytic cleavage during maturation. The molecular mechanisms of capsid assembly and maturation are well understood, but little is known about the viral infectious lifecycle due to a lack of tissue culture cell lines that are susceptible to tetravirus infection. We show here that binding and entry of the alphatetravirus, Helicoverpa armigera stunt virus (HaSV), is triggered by alkaline pH. At pH 9.0, wild-type HaSV virus particles undergo conformational changes that induce membrane-lytic activity and binding to Spodoptera frugiperda Sf9 cells. Binding is followed by entry and infection, with virus replication complexes detected by immunofluorescence microscopy within 2h post-infection and the CP after 12h. HaSV particles produced in S. frugiperda Sf9 cells are infectious. Helicoverpa armigera larval virus biofeed assays showed that pre-treatment with the V-ATPase inhibitor, Bafilomycin A1, resulted in a 50% decrease in larval mortality and stunting, while incubation of virus particles at pH 9.0 prior to infection restored infectivity. Together, these data show that HaSV, and likely other tetraviruses, requires the alkaline environment of the lepidopteran larval midgut for binding and entry into host cells.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O’nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Proteolytic processing of mesonivirus replicase polyproteins by the viral 3C-like protease.

Mesoniviridae are a family of insect RNA viruses that diverged profoundly from other families of the Nidovirales. Mesonivirus replicative proteins are produced from large polyprotein (pp) precursors (pp1a and pp1ab) through proteolytic cleavage by the viral 3C-like protease (3CLpro) and, possibly, other proteases. Using recombinant forms of the Cavally virus 3CLpro and pp1a/pp1ab-derived substrates, we characterized 3CLpro cleavage sites in mesonivirus polyproteins. Our data lead us to suggest that 3CLpro cleaves the central and C-proximal regions of mesonivirus pp1a/pp1ab at 12 conserved sites. Compared to other nidovirus homologues, the mesonivirus 3CLpro features a distinct substrate specificity, with asparagine at P2 being a major specificity determinant. Furthermore, we provide evidence that expression of the ORF1b-encoded part of pp1ab involves a -1 ribosomal frameshift at a conserved GGAUUUU heptanucleotide sequence in the ORF1a/1b overlap region. Taken together, the study identifies critical steps in the expression and maturation of mesonivirus replicative proteins.

Are arthropods at the heart of virus evolution?

The huge diversity of negative-sense RNA viruses in insects, spiders and other arthropods suggests that these animals could be central to virus origin and evolution.

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response.

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.

Wolbachia influences the maternal transmission of the gypsy endogenous retrovirus in Drosophila melanogaster.

ABSTRACTThe endosymbiotic bacteria of the genus Wolbachia are present in most insects and are maternally transmitted through the germline. Moreover, these intracellular bacteria exert antiviral activity against insect RNA viruses, as in Drosophila melanogaster, which could explain the prevalence of Wolbachia bacteria in natural populations. Wolbachia is maternally transmitted in D. melanogaster through a mechanism that involves distribution at the posterior pole of mature oocytes and then incorporation into the pole cells of the embryos. In parallel, maternal transmission of several endogenous retroviruses is well documented in D. melanogaster. Notably, gypsy retrovirus is expressed in permissive follicle cells and transferred to the oocyte and then to the offspring by integrating into their genomes. Here, we show that the presence of Wolbachia wMel reduces the rate of gypsy insertion into the ovo gene. However, the presence of Wolbachia does not modify the expression levels of gypsy RNA and envelope glycoprotein from either permissive or restrictive ovaries. Moreover, Wolbachia affects the pattern of distribution of the retroviral particles and the gypsy envelope protein in permissive follicle cells. Altogether, our results enlarge the knowledge of the antiviral activity of Wolbachia to include reducing the maternal transmission of endogenous retroviruses in D. melanogaster.

Newly discovered insect RNA viruses in China

Insects are a group of arthropods and the largest group of animals on Earth, with over one million species described to date. Like other life forms, insects suffer from viruses that cause disease and death. Viruses that are pathogenic to beneficial insects cause dramatic economic losses on agriculture. In contrast, viruses that are pathogenic to insect pests can be exploited as attractive biological control agents. All of these factors have led to an explosion in the amount of research into insect viruses in recent years, generating impressive quantities of information on the molecular and cellular biology of these viruses. Due to the wide variety of insect viruses, a better understanding of these viruses will expand our overall knowledge of their virology. Here, we review studies of several newly discovered RNA insect viruses in China.

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers.

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.

Membrane targeting of an alpha-like tetravirus replicase is directed by a region within the RNA-dependent RNA polymerase domain

The members of the family Tetraviridae are small positive-sense insect RNA viruses that exhibit stringent host specificity and a high degree of tissue tropism, suggesting that complex virus-host interactions are likely to occur during infection and viral replication. The alpha-like replicase of Helicoverpa armigera stunt virus (HaSV) (genus Omegatetravirus) has been proposed to associate with membranes of the endocytic pathway, which is similar to Semliki Forest virus, Sindbis virus and rubella virus. Here, we have used replicase-EGFP fusion proteins and recombinant baculovirus expression to demonstrate that the HaSV replicase associates strongly with cellular membranes, including detergent-resistant membranes, and that this association is maintained through a novel membrane targeting domain within the C-terminal region of the RNA-dependent RNA polymerase domain. We show a similar subcellular localization and strong association with detergent-resistant membranes for the carmo-like replicase of another tetravirus, Providence virus, in replicating cells, suggesting a common site of replication for these two tetraviruses.

An inhibitory function of WW domain-containing host proteins in RNA virus replication

To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92pol was reduced when Wwm1 was over-expressed, suggesting that the stability of p92pol might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy.