Dataset Information

A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response.

ABSTRACT: 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.

SUBMITTER: Avadhanula V

PROVIDER: S-EPMC2738967 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response.

Avadhanula Vasanthi V Weasner Brandon P BP Hardy Gail G GG Kumar Justin P JP Hardy Richard W RW

PLoS pathogens 20090918 9

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 R ...[more]

PMID: 19763182

Similar Datasets

Project description:Hepatitis B virus (HBV) replicates its genomic DNA by reverse transcription of an RNA intermediate, termed pregenomic RNA (pgRNA), within nucleocapsid. It had been shown that transfection of in vitro-transcribed pgRNA initiated viral replication in human hepatoma cells. We demonstrated here that viral capsids, single-stranded DNA, relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA) became detectable sequentially at 3, 6, 12, and 24 h post-pgRNA transfection into Huh7.5 cells. The levels of viral DNA replication intermediates and cccDNA peaked at 24 and 48 h post-pgRNA transfection, respectively. HBV surface antigen (HBsAg) became detectable in culture medium at day 4 posttransfection. Interestingly, the early robust viral DNA replication and cccDNA synthesis did not depend on the expression of HBV X protein (HBx), whereas HBsAg production was strictly dependent on viral DNA replication and expression of HBx, consistent with the essential role of HBx in the transcriptional activation of cccDNA minichromosomes. While the robust and synchronized HBV replication within 48 h post-pgRNA transfection is particularly suitable for the precise mapping of the HBV replication steps, from capsid assembly to cccDNA formation, targeted by distinct antiviral agents, the treatment of cells starting at 48 h post-pgRNA transfection allows the assessment of antiviral agents on mature nucleocapsid uncoating, cccDNA synthesis, and transcription, as well as viral RNA stability. Moreover, the pgRNA launch system could be used to readily assess the impacts of drug-resistant variants on cccDNA formation and other replication steps in the viral life cycle. IMPORTANCE Hepadnaviral pgRNA not only serves as a template for reverse transcriptional replication of viral DNA but also expresses core protein and DNA polymerase to support viral genome replication and cccDNA synthesis. Not surprisingly, cytoplasmic expression of duck hepatitis B virus pgRNA initiated viral replication leading to infectious virion secretion. However, HBV replication and antiviral mechanism were studied primarily in human hepatoma cells transiently or stably transfected with plasmid-based HBV replicons. The presence of large amounts of transfected HBV DNA or transgenes in cellular chromosomes hampered the robust analyses of HBV replication and cccDNA function. As demonstrated here, the pgRNA launch HBV replication system permits the accurate mapping of antiviral target and investigation of cccDNA biosynthesis and transcription using secreted HBsAg as a convenient quantitative marker. The effect of drug-resistant variants on viral capsid assembly, genome replication, and cccDNA biosynthesis and function can also be assessed using this system.

Project description:UnlabelledAlphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites.ImportanceAlphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

Dataset Information

A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response.

Publications

A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets