The G490E mutation in reverse transcriptase does not impact tRNA primer selection by HIV-1 with altered PBS and...

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcription normally initiates using a specific cellular transfer RNA (tRNA), tRNA(Lys,3).
• The primer binding site (PBS) on the viral genome and the tRNA's 3' end are complementary.
• Previous research indicates HIV-1 can be engineered to use alternative tRNAs by altering the PBS and U5 regions, though reversion to tRNA(Lys,3) is common.

Purpose of the Study:

• To investigate the impact of a specific reverse transcriptase (RT) mutation, G490E, on HIV-1's ability to utilize alternative tRNA primers.
• To determine if the G490E mutation influences primer selection when the U5 region (A-loop or PAS) and PBS are modified to be complementary to alternative tRNAs like tRNA(Trp) or tRNA(Lys1,2).

Main Methods:

• Construction of HIV-1 variants with modified PBS and U5 regions (A-loop or PAS) to be complementary to tRNA(Trp) or tRNA(Lys1,2).
• Introduction of the G490E mutation into the reverse transcriptase (RT) gene of these variants.
• Analysis of viral replication, infectivity, and primer selection in cell cultures (SupT1, PBMCs).

Main Results:

• HIV-1 variants engineered to use tRNA(Trp) with or without the G490E mutation showed no significant differences in infectivity or replication.
• The G490E mutation did not prevent or alter the reversion of tRNA(Trp)-primed viruses to using tRNA(Met).
• In viruses forced to use tRNA(Lys1,2), the G490E mutation also had no discernible effect on infectivity, growth, or viral fitness.

Conclusions:

• HIV-1 can be successfully directed to use alternative tRNA primers (e.g., tRNA(Trp), tRNA(Lys1,2)) through mutations in the U5 region (PAS or A-loop) and PBS.
• The G490E mutation in reverse transcriptase (RT) does not play a significant role in dictating the selection of these alternative primers.
• The viral genome's U5 region and PBS are the primary determinants for alternative primer selection, independent of the RT G490E mutation's influence on fitness.