Experimental design of HiCapR and overall HIV-1 contact matrix

A) Experimental design of HiCapR for profiling RNA-RNA interactions and dynamics of the HIV genome. Based on the simplified SPLASH protocol, HiCapR incorporates a probe-based hybridization method to enrich HIV RNA chimeras from the SPLASH library. The HiCapR method has been applied to two strains of HIV, NL4-3 and GX2005002, in both infected cells and their corresponding virus particles.

B) Comprehensive contact matrix derived from infected cells and virions of both NL4-3 and GX2005002 HIV-1 strains in the HiCapR experimental groups. The heatmap displays the average count of chimeras per 1 million mapped reads, combining data from two biological replicates for each sample.

conformations of HIV-1 5’-UTR

A) Known conformation of HIV-1 5’-UTR supported by chimeras. Previous reported stems in 5’-UTR are supported by chimeras from HiCapR. Colors of the nucleotides indicate the log2 transformed base pairing scores.

B) Representation of the one-dimensional structure of the HIV-1 5’-UTR, highlighting the conservation between the GX2005002 and NL4-3 strains in this region. The diagram includes rectangular boxes denoting the locations of key structural elements, with numerical coordinates referencing NCBI DNA genome coordinates. Dashed boxes indicate regions that are either absent or distinct in GX2005002 compared to NL4-3. Small triangle arrows indicate insertions in the GX2005002 5’-UTR. At the top, a seqlogo displays the consensus nucleotides in the SL1 region.

C) Multidimensional scaling (MDS) plot clustering each of the 1,000 computationally predicted structures of the 5’-UTR in two strains under two conditions.

D) Basepairing probability matrices were calculated from 1000 computationally predicted structures, with color indicating the percentage of structures supporting the specific basepair.

Identification and validation of homodimers in HIV-1 genome

A) Visual depiction of the RNA homodimer formation process: Inter-ligated fragments (arms) originating from homodimeric RNA molecules generate chimeras, where each arm aligns to overlapping coordinates on the HIV-1 genome. This process enables the plotting of coverage and specific details of dimers by utilizing the positions and counts of these overlapping chimeras.

B) distribution of homodimers throughout the HIV-1 genome. The blue line plot showcases the homodimer coverage derived from ligated samples of NL4-3 infected cells along the HIV-1 genome. Arc plots exhibit discontinuous reads in non-ligated samples of NL4-3 infected cells, with dark red segments indicating peaks of homodimers. The lower panels depict the base pairing of homodimers in the SL1 region of the 5’-UTR and downstream of the RRE region. Color mapping indicates the log2 transformed dimer score.

C) Evaluation of the self-binding of dimers using Bio-Layer Interferometry (BLI). The data presented is the result of analysis from three independent experiments, providing insights into the interaction and binding behavior of the dimers.

genome domains along HIV-1 genome

A) Each panel shows triangle matrix and genome domains of two strains of the virus in both cell and virion states, as calculated using C-world. The x-axis represents the genomic position, while the y-axis represents the insulation score (solid lines) and dimer coverage (dashed lines).

B) Correlation of insulation score in infected cells and virion for NL4-3 and GX2005002 strain.

C) Violin and boxplot comparing the boundary strength of the genomic domains of two strains between infected cells and virions. The boxplot displays the median, quartiles, and range of the boundary strength values for each strain in each state.

long range interaction in HIV-1 genome

A) heatmaps of enriched interactions obtained from NL4-3 and GX2005002 infected cells and virions. The upper diagonal shows interactions from infected cells, while the lower diagonal region displays interactions from virions

B) Viewpoint lines depicts the binding positions of the 5’-UTR along the HIV-1 genome. The gray rectangles indicate the viewpoint regions. The colors of the lines represent specific samples, with samples from virions shown as dashed lines.

C) Contact matrices and base pairing details between 5’-UTR and 3’-UTR. The top panels display heatmaps indicating contact probability, with the color bar indicating chimeric reads per 1M reads in each specific sample. The bottom panels show base pairing colored by base pairing scores.