Adenovirus: Invasion of the Cell
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Abstract
Adenovirus is a double-stranded DNA virus with a non-enveloped, icosahedral capsid that causes localized infections, such as conjunctivitis, common cold, and other upper respiratory tract infections. Adenovirus consists of multiple serotypes, different strains that are recognized by different antibodies produced by the immune system. When a virus infects a cell, it takes control of many of the host’s cellular mechanisms, specifically replication and protein synthesis. The protein E4 11k, a product of E4ORF3 gene expression, is an early viral protein that inhibits host cell protein synthesis, controls the expression of late viral genes, and binds to processing body (P-body) proteins, particularly Ddx6. P-bodies are made up of translationally inactive mRNAs and proteins that are involved in mRNA degradation and translational regulation. They are normally found in the cytoplasm in low levels and in elevated levels when the cell is responding to stressors. The goal of our project is to find the binding site of E4 11k to Ddx6, whether as a single nucleotide or a sequence of nucleotides. To accomplish this, HeLa cells will be transfected with hybrids of adenovirus serotypes 5 and 9, and Ddx6 and subjected to co-immunoprecipitation to determine binding abilities. The first step in ensuring high transfection efficiency is using a reagent, such as FuGene 6, to form a complex with DNA in order to transport it into the host cell. It also minimizes cellular toxicity. This assay should help us narrow down the binding site to one of three regions. We can do further site-directed mutagenesis to identify one or a few amino acids involved in binding. Once the binding site is determined, we can evaluate the role of the interaction between Ddx6 and E4 11k on the function of E4 11k in controlling host and viral protein synthesis.
Adenovirus: Invasion of the Cell
Adenovirus is a double-stranded DNA virus with a non-enveloped, icosahedral capsid that causes localized infections, such as conjunctivitis, common cold, and other upper respiratory tract infections. Adenovirus consists of multiple serotypes, different strains that are recognized by different antibodies produced by the immune system. When a virus infects a cell, it takes control of many of the host’s cellular mechanisms, specifically replication and protein synthesis. The protein E4 11k, a product of E4ORF3 gene expression, is an early viral protein that inhibits host cell protein synthesis, controls the expression of late viral genes, and binds to processing body (P-body) proteins, particularly Ddx6. P-bodies are made up of translationally inactive mRNAs and proteins that are involved in mRNA degradation and translational regulation. They are normally found in the cytoplasm in low levels and in elevated levels when the cell is responding to stressors. The goal of our project is to find the binding site of E4 11k to Ddx6, whether as a single nucleotide or a sequence of nucleotides. To accomplish this, HeLa cells will be transfected with hybrids of adenovirus serotypes 5 and 9, and Ddx6 and subjected to co-immunoprecipitation to determine binding abilities. The first step in ensuring high transfection efficiency is using a reagent, such as FuGene 6, to form a complex with DNA in order to transport it into the host cell. It also minimizes cellular toxicity. This assay should help us narrow down the binding site to one of three regions. We can do further site-directed mutagenesis to identify one or a few amino acids involved in binding. Once the binding site is determined, we can evaluate the role of the interaction between Ddx6 and E4 11k on the function of E4 11k in controlling host and viral protein synthesis.