The effect of template RNA structure on elongation by HIV-1 reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Retroviral RNA genomes contain structured regions essential for viral life cycle functions.
• Human immunodeficiency virus type 1 (HIV-1) possesses TAR and polyA hairpin structures in its R region.
• Efficient reverse transcription requires navigating these RNA structures.

Purpose of the Study:

• To investigate the impact of RNA secondary structure stability on HIV-1 reverse transcriptase (RT) elongation.
• To determine how viral nucleocapsid protein (NC) affects RT pausing at structured regions.
• To understand how retroviruses manage RNA structure during reverse transcription.

Main Methods:

• In vitro analysis of HIV-1 RT elongation on wild-type and mutant RNA templates with varying polyA hairpin stability.
• Assessment of pause cDNA products to quantify RT pausing.
• Addition of purified NC protein to in vitro assays to observe its effect on RT pausing.

Main Results:

• Increased RNA secondary structure stability directly correlated with increased RT pausing.
• RT paused approximately 6 nucleotides upstream of hairpin stems, suggesting stalling due to domain contact.
• Viral NC protein effectively overcame structure-induced RT pauses in vitro.
• High Mg2+ concentrations exacerbated pausing by stabilizing RNA structures.
• Structure-induced pausing was not observed in cell-based assays, implying virion factors mitigate the effect.

Conclusions:

• HIV-1 RT encounters significant challenges elongating through stable RNA secondary structures.
• Viral NC protein plays a crucial role in facilitating reverse transcription through structured regions.
• Retroviruses strategically utilize stable RNA structures to regulate viral processes without halting reverse transcription.
• In vivo, virion components likely compensate for the in vitro observed limitations of RT.