Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs

Area of Science:

• Molecular Biology
• Virology
• RNA Structure and Function

Background:

• Tat protein is essential for human immunodeficiency virus type 1 (HIV-1) gene expression.
• Tat binds to the TAR (transactivation response) RNA element, which contains a bulge and a loop.
• In vitro studies show Tat binds the TAR bulge, but in vivo activation requires loop sequences, suggesting a cellular factor interaction.

Purpose of the Study:

• To investigate the functional independence of Tat-TAR RNA interactions.
• To determine if the Tat-binding bulge and the loop-binding cellular factor interaction can be spatially separated.
• To explore the orientation requirements for complementation of mutated TAR elements.

Main Methods:

• Construction of double TAR elements with various combinations of mutated TAR structures.
• Assessment of Tat-mediated gene activation using these engineered TAR elements.
• Analysis of complementation between defective TAR elements with mutations in the bulge or loop.

Main Results:

• Mutated TAR elements with defects in either the bulge or the loop could complement each other to restore wild-type Tat activation.
• This complementation was orientation-dependent, requiring the intact Tat binding site on the 5'-proximal TAR element.
• The results indicate that the bulge and loop interaction sites do not need to be on the same TAR molecule.

Conclusions:

• Tat-TAR RNA activation is a two-component system involving Tat-bulge and cellular factor-loop interactions.
• These two interaction components can function independently and be provided in a trans-acting manner.
• Spatial separation of these components is possible, provided the correct orientation is maintained.