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. 2016 Aug 25;166(5):1257-1268.e12.
doi: 10.1016/j.cell.2016.07.044.

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential During Virion Morphogenesis

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Free PMC article

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential During Virion Morphogenesis

Jacques J Kessl et al. Cell. .
Free PMC article

Abstract

While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

Figures

Figure 1
Figure 1. CLIP-seq experiments showing HIV-1 IN binding to viral RNA in virions
(A) A representative autoradiogram of IN-RNA crosslinked adducts from HIV-1NL4-3 and HIV-1NL4-3 ΔIN virions (upper panel). Corresponding western blot analysis of IN in immunoprecipitated fractions (lower panel). (B) Percentage of reads that map to cellular and viral RNA obtained from 2 independent CLIP-Seq analysis of IN-RNA adducts from WT virions. Error bars indicate standard errors. (C) CLIP-seq results showing read densities (frequency distribution of nucleotide occurrence) mapped to the viral genome obtained from HIV-1NL4-3 (red) and HIV-1NL4-3 ΔIN (blue) progeny virions. The read densities have been normalized with respect to total reads for WT HIV-1NL4-3. A collinear schematic diagram of the HIV-1 genome is shown above.
Figure 2
Figure 2. HIV-1 IN binding to synthetic viral RNA segments in vitro
(A) Schematic for the SEC approach to test IN interactions with nucleic acids. (B) SDS-PAGE and immunoblotting with anti-IN antibodies of SEC fractions. Lane 1: molecular weight markers; Lane 2: 1/2 input IN; Lane 3: unliganded IN subjected to SEC; Lanes 4 and 5: IN+vDNA or IN+vRNA(1-850) complexes were assembled in the buffer containing 0.1 M NaCl and subjected to SEC in the same buffer. Lanes 6 and 7: IN+vDNA or IN+vRNA(1-850) complexes were formed in the buffer containing 0.1 M NaCl. Then NaCl concentration was increased to 1M followed by SEC. (C) AlphaScreen assay design to monitor direct binding between 6xHis-tagged WT IN and biotinylated RNA oligonucleotides (only IN dimer shown for simplicity). “A” and “D” indicate anti-His Acceptor and streptavidin coated Donor beads bound to 6xHis-tagged IN or biotin respectively. (D) Representative AlphaScreen binding curves for IN binding to vRNA(1-57)-TAR in the presence of 0.1 M or 1 M NaCl. The average values of three independent experiments and corresponding standard deviations are shown. (E and F) Summary of AlphaScreen-based assays for IN binding affinities for various RNA oligonucleotides with standard errors of three independent experiments shown.
Figure 3
Figure 3. Biophysical analysis of IN-vRNA(1-850) complexes
AFM images of SEC purified samples: IN alone (A), vRNA(1-850) alone (B) and IN+vRNA(1-850) (C and D). (D) The zoomed image of the box in (C) with arrows pointing to the bright spots corresponding to IN bound to vRNA(1-850). (E) The design of AlphaScreen-based RNA bridging assay (only IN dimer shown for simplicity). “A” and “D” indicate anti-DIG Acceptor and streptavidin coated Donor beads bound to either biotin or digoxin (DIG), respectively. (F) The results of the RNA bridging assay with 25 nM of total RNA incubated with indicated concentrations of IN and RT. The average values of three independent experiments and corresponding standard deviations are shown. (G) BS3 crosslinking to analyze the multimeric state of IN in the context of unliganded IN (lanes 2 and 3), IN+ribonucleotides mixture (rNTPs) (lanes 5 and 6), IN+vRNA(237-279)-DIS (lanes 8 and 9), and IN+vRNA(1-850) (lanes 11 and 12) in vitro compared to the multimeric states of IN in virions of HIV-1NL4-3 (lanes 14 and 15). Positions of monomeric (M) and dimeric (D) forms as well as higher-order aggregates are indicated.
Figure 4
Figure 4. Mapping IN residues binding to vRNA(1-57)-TAR using MS-based protein footprinting
Representative segments of MS spectra showing that two IN peptides 263-269 (containing modified K264 and K266) and 270-284 (containing modified K273) are detected with unliganded IN (middle panels) but are significantly diminished in the IN-vRNA(1-57)-TAR complex (lower panels). In contrast, the peptide 216-224 (containing modified K219) persists with both unliganded IN and the IN-vRNA(1-57)-TAR complex. The unmodified peptides 200-211 and 245-262 serve as internal controls. Shown below is a graphical representation of HIV-1 IN protein with three domains and identified RNA binding residues indicated.
Figure 5
Figure 5. Biochemical characterization of IN mutants
(A) Interactions of recombinant WT, K264A/K266A and R269A/K273A mutant INs with vRNA(1-57)-TAR measured by AlphaScreen. The average values of three independent experiments and corresponding standard deviations are shown. (B) Analytical SEC of WT, K264A/K266A and R269A/K273A mutant INs. Monomeric (M) and tetrameric (T) INs are indicated. (C) Affinity-pull-down assays showing binding of WT and mutant INs to LEDGF/p75. Lane 1: molecular weight markers; Lanes 2–5 loads of 6xHis-tagged WT IN, IN(K264A/K266A), IN(R269A/K273A) and tag-free LEDGF/p75; Lanes 6–9: affinity pull-down using Ni beads of LEDGF/p75 with 6xHis-tagged WT IN, IN(K264A/K266A) and IN(R269A/K273A). (D) Agarose gel analysis of concerted integration products. Lane 1: DNA markers (BIOLINE Quanti-Marker, 1 kb); Lane 2: linearized target DNA; Lanes 4–6: activities of WT, K264A/K266A and R269A/K273A mutant INs in the absence of LEDGF/p75; Lanes 7–9: activities of WT, K264A/K266A and R269A/K273A mutant INs in the presence of LEDGF/p75. Target and donor DNA substrates as well as single-site and concerted integration products are indicated.
Figure 6
Figure 6. Characterization of mutant viruses
(A) Autoradiograms of IN-RNA and NC-RNA crosslinked adducts from WT HIV-1NL4-3, HIV-1NL4-3 IN(K264A/K266A) and HIV-1NL4-3 IN(R269A/K273A) virions (upper two panels). Corresponding western blot analysis of IN and NC in immunoprecipitated fractions (lower two panels). (B) CLIP-seq results for IN-viral RNA crosslinks from WT HIV-1NL4-3, HIV-1NL4-3 IN(K264A/K266A) and HIV-1NL4-3 IN(R269A/K273A) virions (only the first 1000 nucleotides shown). (C) Representative EM images of virions from WT HIV-1NL4-3, HIV-1NL4-3 IN(K264A/K266A) and HIV-1NL4-3 IN(R269A/K273A). (D) Sucrose density gradient separation of capsid cores from detergent-lysed virions of WT HIV-1NL4-3, HIV-1NL4-3 IN(K264A/K266A) and HIV-1NL4-3 IN(R269A/K273A). (E) Quantitation of triplicate experiments of HIV-1 CA-p24 signal intensity from (D) using ImageJ software. The error bars indicate standard deviations.
Figure 7
Figure 7. Effects of BI-B2 on HIV-1 IN interactions with viral RNA in virions
(A) Virions from WT HIV-1NL4-3 and the escape mutant HIV-1NL4-3 IN(A128T) were prepared in the presence and absence of 10 μM BI-B2 in virus producer cells and used to infect target cells to determine relative infectivity. The average values of three independent experiments and corresponding standard deviations are shown. (B) Autoradiograms of IN-RNA and NC-RNA crosslinks adducts from virions of WT HIV-1NL4-3 and HIV-1NL4-3 IN(A128T) prepared in the presence and absence of 10 μM BI-B2 (upper two panels). Corresponding western blot analysis of IN and NC in immunoprecipitated fractions (lower two panels). (C) CLIP-seq analysis of IN binding to viral RNA genome from virions of WT HIV-1NL4-3 (upper panel), WT HIV-1NL4-3 + BI-B2 (middle panels), and HIV-1NL4-3 IN(A128T) + BI-B2 (lower panel).

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