Inosine-Induced Base Pairing Diversity during Reverse Transcription.

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Adenosine to inosine (A-to-I) editing is crucial for cellular functions and RNA virus infectivity.
• Inosine modifications can alter viral RNA replication and cellular transcriptomes, resembling A-G transitions.
• The impact of hyper-editing on viral polymerase activity and sequencing fidelity remains unclear.

Purpose of the Study:

• To investigate the influence of inosine modification number and position on reverse transcriptase (RT) decoding outcomes.
• To elucidate the structural basis of inosine accommodation in HIV-1 RT and its effect on base pairing.
• To understand how hyper-editing affects RNA sequencing and transcription.

Main Methods:

• Systematic exploration of inosine modification effects on decoding using three different RTs and Sanger sequencing.
• Molecular dynamics simulations of HIV-1 RT to analyze inosine accommodation and base pairing.
• Varying the number and position of inosines in RNA substrates.

Main Results:

• Inosine modifications, individually or in clusters, differentially affect RT activity.
• Molecular dynamics simulations revealed that protein-nucleotide interactions are key to understanding inosine cluster base pairing in HIV-1 RT.
• Sanger sequencing results can be reshaped by hypermodified inosines during reverse transcription.

Conclusions:

• Inosine hyper-editing significantly impacts RT fidelity and decoding, potentially altering RNA sequencing outcomes.
• Structural insights into HIV-1 RT-inosine interactions are critical for understanding the physiological significance of A-to-I editing.
• This work lays the foundation for decrypting the role of hyper-edited inosines in cellular and viral RNA transcription.