Fig 1.
Cells are fed with the ribonucleoside analog 4-thiouridine (4SU) which is incorporated into nascent RNA. Live cells are then irradiated with ultraviolet (UV) light, which induces covalent cross-links between proteins and RNA at sites of contact and 4SU incorporation. Cells are then lysed and treated with RNase A to generate oligonucleotides crosslinked to proteins of interest. Protein-RNA complexes are then immunopurified, and then the RNA is dephosphorylated at the 3'-end with alkaline phosphatase. The RNA is subsequently radiolabeled with 32P using T4 polynucleotide kinase (PNK) for detection by autoradiography. Protein-RNA complexes are then separated by SDS-PAGE, transferred to nitrocellulose and a region corresponding to protein-RNA adducts is excised. Cross-linked RNA is isolated by proteinase K treatment and phenol:chloroform extraction. After sequential adapter ligations, the RNA library is reverse transcribed. Reverse transcriptase often misincorporates a G opposite the 4SU cross-linking site, which leads to T to C substitutions in positive-strand of the cDNA, enabling the precise mapping of protein-RNA interaction sites. After PCR amplification, the cDNA library is sequenced by Illumina sequencing.
Fig 2.
Viral RNA binding by A3F, A3G and A3H in cells and mature virions.
(A) A3-RNA cross-linked complexes were immunoprecipitated from mock infected or HIV-1NL4-3 ΔVif infected HEK 293T cells stably expressing 3×HA-tagged A3F, A3G or A3H proteins that had been fed with 4SU and UV-irradiated. A3-RNA cross-linked complexes were also immunoprecipitated from purified HIV-1NL4-3 ΔVif virions that were derived from the 4SU-fed infected cells. Complexes were visualized by autoradiography (top) and Western blot analysis using a polyclonal anti-HA (bottom). (B) Frequency distribution of nucleotide occurrence (read density) in reads that were mapped to the HIV-1NL4-3 genome (left). A colinear schematic diagram of the HIV-1 genome is presented above. Correlation analyses (see methods) of A3 binding to viral RNA in cells from two independent CLIP experiments are shown (right).
Fig 3.
Comparison of A3F, A3G and A3H RNA binding in cells and mature virions.
(A) Comparison of read density frequency distributions on the HIV-1NL4-3 genome for CLIP experiments in which the same A3 protein was immunoprecipitated from infected cells and mature virions (left). Correlation analyses of A3 binding frequencies across the viral RNA genome in infected cells and mature virions (right). (B) Comparisons of read density frequency distributions on the HIV-1NL4-3 genome for CLIP experiments in which different A3 proteins were immunoprecipitated from infected cells or mature virions (left). Correlation analyses of different A3 protein binding frequencies across the viral RNA genome in infected cells or mature virions (right).
Fig 4.
RNA targets of A3F, A3G and A3H in cells and mature virions.
(A) Percentages of reads derived from the human and HIV-1 genomes in RNA-seq and A3-CLIP experiments performed using infected cells. Reads were normalized by cellular mRNA frequencies. (B) Origins of individual reads that map to the human and HIV-1NL4-3 genomes in A3 CLIP experiments with uninfected cells, infected cells and purified mature virions. Numbers below each chart are the total number of mapped reads.
Fig 5.
Nucleotide composition of preferred A3 protein binding sites.
(A) The 100 PARalyzer-generated clusters in mRNAs with the greatest number of total associated reads in A3 CLIP experiments were inspected and the fraction the sequence composed of each of the four nucleotides was plotted (top). PARalyzer-generated clusters were filtered according to size (<50 nucleotides) and the clusters with the greatest number of total associated reads in A3 CLIP experiments were inspected and the fraction the sequence composed of each of the four nucleotides was plotted (bottom). (B) Deoxyribonucleotide sequence motifs identified by cERMIT in CLIP-seq cDNA libraries as most frequently present in A3-bound RNA in uninfected cells, infected cells and in immature and mature purified virions. Cumulative percentages of clusters containing the motifs are indicated.
Fig 6.
Interplay between A3, Gag and NC binding to HIV-1 RNA in infected cells or mature virions.
(A) Comparisons of read density frequency distribution on the HIV-1NL4-3 genome for CLIP experiments in which A3 or Gag were immunoprecipitated from infected cells (left). Correlation analyses of A3 and Gag binding frequencies across the viral RNA genome in infected cells (right). (B) Comparisons of read density frequency distributions on the HIV-1NL4-3 genome for CLIP experiments in which A3 and NC were immunoprecipitated from purified mature virions (left). Correlation analyses of A3 and NC binding frequencies across the viral RNA genome (right).
Fig 7.
Interplay between A3 and Gag RNA binding to HIV-1 RNA in immature virions.
(A) A3-RNA cross-linked complexes were immunoprecipitated from purified immature (PR defective) HIV-1NL4-3 ΔVif virions derived from proviral plasmid-transfected HEK 293T cells stably expressing 3×HA-tagged A3F, A3G or A3H proteins. Complexes were visualized by autoradiography (top) and simultaneous Western blot analyses using anti-HA and anti-Gag antibodies (bottom). Asterisks indicate the presence of IgG heavy and light bands in the immunopreciptated fractions. (B) Comparisons of read density frequency distributions on the HIV-1NL4-3 genome for CLIP experiments in which A3 was immunoprecipitated from purified mature or immature virions (left). Correlation analyses of A3 binding frequencies across the viral RNA genome in mature and immature virions (right). (C) Comparisons of read density frequency distributions on the HIV-1NL4-3 genome for CLIP experiments in which A3 or Gag was immunoprecipitated from immature virions (left). Correlation analyses of A3 and Gag binding frequencies across the viral RNA genome in immature virions (right).
Fig 8.
7SL1 RNA binding by A3, Gag and NC in cells, immature virions and mature virions.
Comparisons of read density frequency distributions on the 7SL1 RNA for CLIP experiments in which A3G, Gag or NC were immunoprecipitated from cells, immature virions and mature virions respectively.