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Endogenous MicroRNA Activity Is Not Affected Following Infection With MicroRNA Controlled Adenovirus

ABSTRACT: Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines, however uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus, adenovirus, we have engineered a hepatocyte-safe wild type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this we have included binding sites for hepatocyte-selective microRNA122a within the 3’ UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of microRNA122a binding sites caused up to 80 fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild type Ad5 (5 x 1010 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild type virus retained full activity within cancer cells and provides a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA-control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

ORGANISM(S): Mus musculus

PROVIDER: GSE23854 | GEO | 2011/08/20

SECONDARY ACCESSION(S): PRJNA130559

REPOSITORIES: GEO

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Project description:Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines, however uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus, adenovirus, we have engineered a hepatocyte-safe wild type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this we have included binding sites for hepatocyte-selective microRNA122a within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of microRNA122a binding sites caused up to 80 fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild type Ad5 (5 x 1010 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild type virus retained full activity within cancer cells and provides a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA-control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases. four groups which we had were: WT: RNA extracted from livers of mice treated with wild type Ad5, 3 Replicates miR: RNA extracted from livers of mice treated with wild type Ad5 bearing mir122-binding sites in the 3'UTR of E1A (3 Replicates) Luc: RNA extracted from livers of mice treated with non-replicating Ad5 (3 Replicates) PBS: RNA extracted from livers of mice treated with PBS only (3 Replicates)

Project description:Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. One way in which host antiviral proteins antagonize viral infection is by associating with viral genomes and inhibiting essential viral processes. Adenovirus (AdV) encodes gene products from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteins manipulated by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, un-biased proteomics approach to analyze changes to the host proteome during infection with Adenovirus serotype 5 (Ad5) virus. We combined quantification of total protein abundance in the proteome together with an analysis of proteins associated with viral genomes using isolation of Proteins on Nascent DNA (iPOND). By Integrating these proteomics datasets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. Importance Viral infections stimulate myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, modifications, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus serotype 5 (Ad5) encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We use an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner that could overcome potentially inhibitory host defenses. This study provides a global view of Adenovirus-mediated proteome remodeling which can serve as a model to investigate virus-host interactions of DNA viruses.

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Endogenous MicroRNA Activity Is Not Affected Following Infection With MicroRNA Controlled Adenovirus

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