Second generation imaging of nuclear/cytoplasmic HIV-1 complexes

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Visualizing fluorescent human immunodeficiency virus type 1 (HIV-1) particles in infected cell nuclei aids in studying viral nuclear biology.
• A previously developed microscopy-based fluorescent system (HIV-IN-EGFP) enabled visualization of HIV-1 complexes and nuclear import efficiency.

Purpose of the Study:

• To enhance the HIV-1 visualization system for improved infectivity and clearer nuclear import studies.
• To analyze HIV-1 nuclear import in cells expressing the TRIMCyp restriction factor using the HIV-IN-EGFP system.

Main Methods:

• Development of a second-generation fluorescent HIV visualization system (HIV-IN(K)sfGFP_IN(E)) using mutated integrase (IN) proteins.
• Utilizing confocal microscopy to analyze viral complexes and nuclear entry.
• Assessing viral infectivity and properties of the modified viral particles.

Main Results:

• The initial HIV-IN-EGFP system showed reduced infectivity due to Vpr-mediated trans-incorporation.
• Mutated IN forms (E11K and K186E) stabilized IN complexes, restoring infectivity to nearly 30% of wild-type.
• The second-generation system (HIV-IN(K)sfGFP_IN(E)) produced smaller fluorescent particles, requiring brighter fluorophores but preserving nuclear entry and enhancing infectivity.

Conclusions:

• The optimized fluorescent HIV visualization system (HIV-IN(K)sfGFP_IN(E)) allows direct visualization of HIV-1 nuclear entry.
• This improved system maintains intact viral properties, including enhanced infectivity.
• The system is valuable for studying HIV-1 nuclear biology and viral events during nuclear import.