The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• EBER-associated protein (EAP) is a cellular RNA-binding protein.
• EAP associates with small RNAs from certain herpesviruses, including Epstein-Barr virus (EBV) and herpesvirus papio (HVP).

Purpose of the Study:

• To characterize the interaction between EAP and EBV-encoded RNA 1 (EBER 1) and HVP 1.
• To determine the binding site and specificity of EAP on EBER 1.

Main Methods:

• Generation of polyclonal anti-EAP antibodies.
• RNA immunoprecipitation assays to identify EAP-associated RNAs.
• RNase protection assays and mutational analyses to map EAP binding sites on EBER 1.
• Binding studies with bacterially expressed fusion proteins.

Main Results:

• EAP associates with over 95% of EBER 1 and the majority of HVP 1 in infected cells.
• EAP directly binds to stem-loop 3 of EBER 1 in a sequence-specific manner, recognizing both single-stranded and double-stranded regions.
• EAP binding is independent of other cellular or viral proteins.
• A potential weaker binding site for EAP may exist on stem-loop 4 of EBER 1.

Conclusions:

• EAP is a key protein involved in the function of EBER 1 and HVP 1.
• The sequence-specific binding of EAP to conserved stem-loop structures is critical for the function of these viral RNAs.