Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the 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 degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the 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 degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

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