HIV Integration

 

HIV infection continues to be a worldwide concern. As a retrovirus, HIV is defined by its reverse transcriptase (RT) and integrase (IN) activities. Both of these enzymes are current targets of anti-retroviral therapies (ART). However, the highly mutagenic nature of the RT frequently results in ART resistance mutations underlining the need for novel drugs and drug targets.

 

Stable integration of HIV into the genome of a host cell is required for a productive infection. HIV integration is mediated by a poorly understood pre-integration complex (PIC), which includes the viral proteins RT, IN, viral protein R (Vpr), and matrix protein (encoded by gag). Several host co-factors that affect HIV integration have been identified.

 

The precise mechanism of HIV integration in vivo remains elusive. We have constructed a single molecule total internal reflection fluorescence (smTIRF) and single molecule magnetic tweezers (smMT) microscopy capable of visualizing the kinetic processes associated with retroviral integration in real time. The smTIRF system is similar to that developed for the mismatch repair studies (see Mismatch Repair Section). Visualization of DNA binding kinetics, the integration site search process and the kinetics of integration as indicated by DNA breakage may be examined in real-time with smTIRF. The smMT system uses a flow cell that tethers DNA to a glass surface by biotin-streptavidin linkage and to a super paramagnetic bead by digoxygenine-antidigoxygenin linkage. Changing the vertical position of two ferromagnets relative to the sample generates a stretching force. The DNA molecule length is determined by gauging the vertical position of the magnetic bead relative to a bead glued to the glass surface. The force on the bead is determined from the bead fluctuations parallel to the glass surface. Ferromagnets are mounted on a stepper motor such that forward and reverse forces may be examined. Quantitative applied forces between 0.1 and 30 piconewtons (pN) are possible with this system and changes in DNA supercoiling or DNA length may be examined in real time.

 

As an example and in collaboration with Dr. Kristine Yoder (Molecular Virology, Immunology and Medical Genetics) we have been begun to examine the DNA binding and site search kinetics of the Prototype Foamy Virus (PFV) retroviral integrase (Figure 1). The system uses smTIRF as diagramed in Figure 1A. Synthetic long terminal repeat (LTR) oligonucleotides labeled with Cy3 are bound by the PFV intergase allowing visualization of the complex. For these studies we use a mutant form of the PFV integrase [PFV(D128N)] that is unable to perform the strand transfer event. Thus, we uniquely focus on the DNA binding and searching process without interference by the actual strand transfer event.

 


Figure 1.   PFV Integration into 24kb λ-DNA.  (A) A biotinylated-PEG surface is applied to a quartz slide, and 24kb λ-DNA is bound to the surface using neutravadin-biotin interactions.  PFV intasomes are then introduced to the system and the reaction is viewed on an smTIRF set up.  (B) λ-DNA is visualized using cytox dye following the experiment.  (C) At an initial time, t = 0, no PFV binding activity is observed on the λ-DNA.  At 5 seconds PFV is observed binding to the λ-DNA.  (D) By determining the dwell times of individual PFV integration events, we can quantitate the lifetime of PFV binding to λ-DNA by fitting the total number of molecules to a single exponential decay.  For 24kb λ-DNA, we have observed an average lifetime of 18.6s.
HIV Int Figure Final r2

 

Questions:

 

1.) Can the smTIRF and smMT systems be developed to examine multiple integration events?

Retroviral integration in vitro is extremely inefficient. This means that under the best of conditions only ~10% of target molecules enjoy an integration event. This makes quantitative kinetic analysis difficult at best. However, if one could develop a system capable of tracking 50 or more molecules simultaneously, then true quantitiative kinetic analysis becomes possible.

 

2.) What are the kinetic and functional processes associated with the two-step retroviral integration reaction?

With the development of the smTIRF and smMT systems will allow the visualization of single integration events. The ability to distinguish single integration events has a number of advantages since the range in the kinetic efficiency may be quantified. In addition, the range of DNA substrates, from naked linear DNA to supercoiled DNA to chromatin made up of defined nucleosomes, may be examined for kinetic efficiency.

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