Sassan Asgari

Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene.

Polydnaviruses produced by the hymenopteran endoparasitoid Cotesia rubecula are deposited together with the egg into the lepidopteran host Pieris rapae and are apparently involved in the suppression of the hosts defence system. Around 6 h post-parasitization host haemocytes change their surface properties, actin cytoskeleton structure and adhesion properties. Here we show that a single polydnavirus gene is expressed inside the caterpillar haemocytes in a transient fashion between 4 to 8 h post-parasitization.

Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.

Endoparasitoids introduce a variety of factors into their host during oviposition to ensure successful parasitism. These include ovarian and venom fluids that may be accompanied by viruses and virus-like particles. An overwhelming number of venom components are enzymes with similarities to insect metabolic enzymes, suggesting their recruitment for expression in venom glands with modified functions. Other components include protease inhibitors, paralytic factors, and constituents that facilitate/enhance entry and expression of genes from symbiotic viruses or virus-like particles. In addition, the venom gland may itself support replication/production of some viruses or virus-like entities. Overlapping functions and structural similarities of some venom, ovarian, and virus-encoded proteins suggest coevolution of molecules recruited by endoparasitoids to maintain their fitness relative to their host.

A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph

Activation of prophenoloxidase (proPO) in insects is a defense mechanism against intruding microorganisms and parasites. Pattern recognition molecules induce activation of an enzymatic cascade involving serine proteinases, which leads to the conversion of proPO to active phenoloxidase (PO). Phenolic compounds produced by pPO-activation are toxic to invaders. Here, we describe the isolation of a venom protein from the parasitoid, Cotesia rubecula, injected into the host, Pieris rapae, which is homologous to serine proteinase homologs (SPH). The data presented here indicate that the protein interferes with the proteolytic cascade, which under normal circumstances leads to the activation of proPO and melanin formation.

West Nile virus encodes a microRNA-like small RNA in the 3′ untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3′ untranslated region (3′-UTR) of the flavivirus genome, in particular the terminal 3′ stem–loop (3′SL) fulfils multiple functions in virus replication and virus–host interactions. Using the Kunjin strain of WNV (WNVKUN), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3′SL. Transcription of WNVKUN pre-miRNA (3′SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNVKUN virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.

Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase, contributing to dengue virus inhibition in Aedes aegypti.

The endosymbiont Wolbachia is common among insects and known for the reproductive manipulations it exerts on hosts as well as inhibition of virus replication in their hosts. Recently, we showed that Wolbachia uses host microRNAs to manipulate host gene expression for its efficient maintenance in the dengue mosquito vector, Aedes aegypti. Cytosine methylation is mediated by a group of proteins called DNA (cytosine-5) methyltransferases, which are structurally and functionally conserved from prokaryotes to eukaryotes. The biological functions of cytosine methylation include host defense, genome stability, gene regulation, developmental promotion of organs, and lifespan regulation. Ae. aegypti has only one DNA methyltransferase gene (AaDnmt2) belonging to the cytosine methyltransferase family 2, which is the most deeply conserved and widely distributed gene among metazoans. Here, we show that in mosquitoes the introduced endosymbiont, Wolbachia, significantly suppresses expression of AaDnmt2, but dengue virus induces expression of AaDnmt2. Interestingly, we found that aae-miR-2940 microRNA, which is exclusively expressed in Wolbachia-infected mosquitoes, down-regulates the expression of AaDnmt2. Reversely, overexpression of AaDnmt2 in mosquito cells led to inhibition of Wolbachia replication, but significantly promoted replication of dengue virus, suggesting a causal link between this Wolbachia manipulation and the blocking of dengue replication in Wolbachia-infected mosquitoes. In addition, our findings provide an explanation for hypomethylation of the genome in Wolbachia-infected Ae. aegypti.

Virus or not? Phylogenetics of polydnaviruses and their wasp carriers.

Our current, still limited, understanding of the comparative biology and evolution of polydnaviruses (PDVs) is reviewed, especially in the context of the possible origins of these parasitoid viruses and of their coevolution with carrier wasps. A hypothetical scenario of evolution of PDVs from ascovirus (or ascovirus-like) ancestors is presented, with examples of apparent extant transitional forms. PDVs appear, in the case of bracoviruses, to show phylogenetic relationships that mirror those of their wasp carriers: with ichnoviruses, the picture is less clear. Ongoing sequencing studies of entire PDV genomes from diverse wasp species are likely to greatly contribute to our understanding of PDV evolution.

A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor.

The molecular mechanism by which polydnaviruses of endoparasitoid wasps disrupt cell-mediated encapsulation reactions of host insects is largely unknown. Here we show that a polydnavirus-encoded protein, produced from baculovirus and plasmid expression vectors, prevents cell surface exposure of lectin-binding sites and microparticle formation during immune stimulation of haemocytes. The inactivation of immune-related cellular processes by this protein was analysed using a specific lectin and annexin V and shown to be virtually identical to polydnavirus-mediated effects on haemocytes. Cytochalasin D application has similar effects on haemocytes, suggesting that the immune suppression by the polydnavirus protein is caused by the destabilization of actin filaments. Since the exposure of cell surface glycoproteins and the formation of microparticles are part of an immune response to foreign objects or microorganisms and a prerequisite for cell-mediated encapsulation of microorganisms and parasites, the virus-encoded protein may become an important tool for the inactivation of cellular immune reactions in insects and an essential component in understanding immune suppression in parasitized host insects.

MicroRNA functions in insects

MicroRNAs (miRNAs) are small non-coding RNAs that are generated in all eukaryotes and viruses. Their role as master regulators of gene expression in various biological processes has only been fully appreciated over the last decade. Accumulating evidence suggests that alterations in the expression of miRNAs may lead to disorders, including developmental defects, diseases and cancer. Here, I review what is currently known about miRNA functions in insects to provide an insight into their diverse roles in insect biology.

An Insect Virus-Encoded MicroRNA Regulates Viral Replication

ABSTRACT MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding RNAs which play an essential role in gene regulation and affect a wide range of processes, including development, differentiation, and oncogenesis. Here we report the identification of the first miRNA from an insect virus, derived from the major capsid protein (MCP) gene in Heliothis virescens ascovirus (HvAV) (HvAV-miR-1). Although MCP was abundantly expressed at all time points 24 h after infection, HvAV-miR-1 expression was strictly regulated and specifically detected from 96 h postinfection. HvAV-miR-1 expression coincided with a marked reduction of the expression of HvAV DNA polymerase I, which is a predicted target. Ectopic expression of full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs transcriptional degradation of DNA polymerase I and regulates HvAV replication. These findings are congruent with recent reports that miR-BART-2 regulates Epstein-Barr virus DNA polymerase expression and suggest that virus-encoded miRNA regulation of virus replication may be a general phenomenon.

Effect of Wolbachia on replication of West Nile virus in a mosquito cell line and adult mosquitoes.

ABSTRACT Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.