Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

Abstract

Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.

Fig 1. RNA sequences used.

HIV-1 genomic RNA partial sequence constructs used in this work. (A) TARpolyA RNA. (B) ΨRNA.

Fig 2. Calibration of AFM cantilever tip.

AFM tip resolution calibration with 2nm diameter Au Sphere. (A) A typical AFM image of 2nm Au sphere on mica in tapping mode in liquid. The scan size is 250nm×250nm. The height color bar scale is 2.5nm. (B) Histogram of 2nm diameter Au sphere for the measured size on the plane of the substrate (top) and the measured height (bottom). Shown in red are normal distribution fits to the peaks. The mean measured size on the plane of the substrate is 7.56 ± 0.09 nm, and the mean height is 2.10 ± 0.02nm. The height is consistent with the sphere diameter. The size in the plane of the substrate reflects the role of the tip size. Please see text and supplemental materials section for more details. The total number of samples was 419 and the experiment was repeated twice.

Fig 3. ΨRNA size and morphology with AFM.

0.5μM ΨRNA on positively charged mica(+). (A) A typical AFM image with a scan size of 500nm×500nm. The height color bar scale is 2.0 nm. A few characteristic ΨRNAs are shown in boxes: monomer (red) and dimer (green). (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histogram, where red arrows indicate monomer and dimer. The mean height is 1.10 ± 0.01nm. The first peak with a mean length of 17.9 ± 0.2nm and width 1.01 ± 0.02nm corresponds to the ΨRNA monomer. The second peak with a mean length of 34.6 ± 0.3nm and width 3.8 ± 0.1nm corresponds to the ΨRNA dimer. The total number of samples was 551 and the experiment was repeated twice.

Fig 4. TARpolyA RNA size and morphology with AFM.

0.5μM TARpolyA RNA on positively charged mica(+). (A) AFM image with a scan size of 500nm×500nm. The height color bar scale is 2.0nm. A few characteristic TARpolyA RNAs are boxed in red. (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks for length and height. Width is fit to a gamma distribution due to its non-negativity and skewness. (C) Three dimensional smooth histogram, where the red arrow indicates the monomer distribution. The peak corresponding to the TARpolyA RNA monomer has a mean length of 17.1 ± 0.2nm, width of 1.10 ± 0.05nm and height of 1.10 ± 0.01nm. The total number of samples was 504 and the experiment was repeated twice.

Fig 5. GagΔP6 size and morphology with AFM.

0.5μM GagΔP6 on negatively charged mica(-). (A) A typical AFM image with a scan size of 500nm×500nm. The height color bar scale is 2.5nm. A few characteristic GagΔP6s are boxed: monomer (red), dimer (green) and tetramer (blue). (B) Histograms for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histogram, where red arrows indicate the monomer, dimer, and tetramer distributions. The mean height is 1.93 ± 0.01nm for all three. The first peak with a mean length of 10.3 ± 0.1nm and width 6.2 ± 0.1nm corresponds to the GagΔP6 monomer. The second peak with a mean length of 20.0 ± 0.2nm and width 6.2 ± 0.1nm corresponds to the GagΔP6 dimer. The third peak with a mean length of 29.0 ± 0.3nm and width 12.9 ± 0.2nm corresponds to the GagΔP6 tetramer. The total number of samples was 858 and the experiment was repeated twice.

Fig 6. Model of GagΔP6 morphology.

Rough model of the GagΔP6 monomer based on the measured mean values of the length and width. MA domain is in red, CA domain is in yellow, and NC domain is in green.

Fig 7. GagΔP6-ΨRNA interaction complex size and morphology.

The mixture of GagΔP6-ΨRNA (0.5μM : 0.5μM) complex on negatively charged mica(-). (A) A typical AFM image with a scan size of 500 nm×500 nm. The height color bar scale is 2.5nm. A few characteristic GagΔP6-ΨRNA complexes are boxed: monomer (red), dimer (green) and tetramer (blue). (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histogram, where red arrows indicate monomer, dimer, and tetramer. The mean height is 1.90 ± 0.01nm for all three complexes. The first peak with a mean length of 10.6 ± 0.2nm and width 6.8 ± 0.1nm corresponds to the GagΔP6-ΨRNA monomer. The second peak with a mean length of 22.4 ± 0.1nm and width 6.8 ± 0.1nm corresponds to the GagΔP6-ΨRNA dimer. The third peak with a mean length of 31.2 ± 0.2nm and width 13.8 ± 0.1nm corresponds to the GagΔP6-ΨRNA tetramer. The total number of samples was 895 and the experiment was repeated twice.

Fig 8. GagΔP6-TARpolyA RNA interaction complex size and morphology.

Mixture of GagΔP6-TARpolyA RNA (0.5μM : 0.5μM) complex on negatively charged mica(-). (A) A typical AFM image with a scan size of 500nm×500nm. The height color bar scale is 2.5nm. A few characteristics TARpolyA RNA complexes are boxed: monomer (red), dimer (green) and tetramer (blue). (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histogram, where monomer, dimer, and tetramer are indicated by red arrows. The mean height is 1.93 ± 0.01nm for all three complexes. The first peak with a mean length of 10.8 ± 0.1nm and width 6.2 ± 0.1nm corresponds to the GagΔP6-TARpolyA RNA monomer. The second peak with a mean length of 20.7 ± 0.2nm and width 6.2 ± 0.1nm corresponds to the GagΔP6-TARpolyA RNA dimer. The third peak with a mean length of 29.9 ± 0.3nm and width 13.6 ± 0.2nm corresponds to the GagΔP6-TARpolyA RNA tetramer. The total number of samples was 766 and the experiment was repeated twice.

Fig 9. GagΔP6-PI(4,5)P2 interaction complex size and morphology.

Mixture of GagΔP6-PI(4,5)P2 (0.5μM : 0.5μM) complex on negatively charged mica(-). (A) A typical AFM image with a scan size of 500nm×500nm. The height the color bar scale is 2.5nm. A few characteristic GagΔP6-PI(4,5)P2 complexes are boxed: monomer (red), dimer (green) and tetramer (blue). (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histogram, where monomer, dimer, and tetramer are indicated by red arrows. The mean height is 1.93 ± 0.01nm for all complexes. The first peak with a mean length of 11.2 ± 0.1nm and width 6.7 ± 0.1nm corresponds to the GagΔP6-PI(4,5)P2 monomer. The second peak with a mean length of 21.1 ± 0.1nm and width 6.7 ± 0.1nm corresponds to the GagΔP6-PI(4,5)P2 dimer. The third peak with a mean length of 30.4 ± 0.2nm and width 14.0 ± 0.2nm corresponds to the GagΔP6-PI(4,5)P2 tetramer. The total number of samples was 903 and the experiment was repeated twice.

Fig 10. PI(4,5)P2-ΨRNA-GagΔP6 interaction complex size and, morphology.

Mixture of PI(4,5)P2-ΨRNA-GagΔP6 (0.5μM : 0.5μM : 0.5μM) complex on negatively charged mica(-). (A) A typical AFM image with a scan size of 500nm×500nm. The height color bar scale is 2.5nm. A few characteristic PI(4,5)P2-ΨRNA-GagΔP6 complexes are boxed: monomer (red), dimer (green) and tetramer (blue). (B) Histogram for length (left), width (middle), and height (right). Shown in red are normal distribution fits to the peaks. (C) Three dimensional smooth histograms, where monomer, dimer, and tetramer are indicated by red arrows. The mean height is 1.91 ± 0.01nm for all three complexes. The first peak with a mean length of 10.9 ± 0.3nm and width 7.4 ± 0.1nm corresponds to the PI(4,5)P2-ΨRNA-GagΔP6 monomer. The second peak with a mean length of 23.8 ± 0.2nm and width 7.4 ± 0.1nm corresponds to the PI(4,5)P2-ΨRNA-GagΔP6 dimer. The third peak with a mean length of 23.8 ± 0.2nm and width 22.1 ± 0.2nm corresponds to the PI(4,5)P2-ΨRNA-GagΔP6 tetramer. The total number of samples was 616 and the experiment was repeated twice.

Fig 11. Model of PI(4,5)P2-ΨRNA-GagΔP6 interaction complex.

Rough models of PI(4,5)P2-ΨRNA-GagΔP6 complexes based on the measured mean height and width. (a) Dimer complex, (b) Tetramer complex. MA domain is in red, CA domain is in yellow, NC domain is in green, ΨRNA is in cyan, and PI(4,5)P2 is in purple.