Mechanisms that prevent template inactivation by HIV-1 reverse transcriptase RNase H cleavages

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses RNase H activity essential for viral replication, cleaving the RNA genome during DNA synthesis.
• Previous studies identified RT-mediated pausing and RNase H cleavage at hairpin structures in RNA templates, generating 3' end-directed cuts.

Purpose of the Study:

• To investigate the correlation between RT-mediated pausing and RNase H cleavage products.
• To determine the impact of secondary cuts on DNA primer extension and viral replication.
• To explore the role of nucleocapsid protein (NC) and dNTPs in modulating RNase H activity.

Main Methods:

• Utilized a hairpin-containing RNA template system to analyze primer extension and RNase H cleavage by HIV-1 RT.
• Quantified and characterized cleavage products generated during synthesis, including those induced by pausing and stalled synthesis.
• Assessed the effect of varying dNTP concentrations and the addition of NC on pausing and cleavage events.

Main Results:

• Prominent RNase H cleavage products were directly correlated with RT-induced pausing at the hairpin base.
• Persistent secondary cleavage products, occurring approximately eight nucleotides from the primer terminus, reduced primer extendibility.
• Increased dNTP concentration or the addition of NC significantly decreased pausing and secondary cut generation.
• 3'-azido-3'-deoxythymidine-induced stalling also resulted in 3' end-directed cleavages, potentially contributing to drug inhibition.

Conclusions:

• RT-mediated pausing at hairpin structures leads to detrimental RNase H cleavages that can impede DNA synthesis.
• Secondary cuts reduce primer extendibility, providing a mechanism for their persistence.
• Factors like NC and optimal dNTP levels enhance DNA synthesis by suppressing these inhibitory RNase H cleavages.
• These findings highlight the importance of factors enhancing DNA synthesis in preventing RNase H activity detrimental to HIV-1 replication.