Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid

Abstract

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

Keywords: CPSF6; HIV; TEM; capsid; imaging; restriction.

FIG 1

Purification of CPSF6-358 with an albumin tag from the mammalian secretory expression system. (A) SDS-PAGE and Western blot analysis of His₆-albumin–CPSF6-358 expression and purification. Samples taken from untransfected cells (U), transfected cells (T), the flowthrough (FT) and elution (E) from Ni-NTA resin, and peaks (P1 and P2) from the Superdex 200 26/60 column (shown in panel B) were stained with Coomassie blue (top) or processed with anti-His (middle) or anti-CPSF6 (bottom) antibody, following Western blotting. (B) Gel filtration profile of the protein eluted from the Superdex 200 26/60 column. The two His₆-albumin–CPSF6-358 peaks are labeled P1 and P2. (C) Representative EM images of negatively stained His₆-albumin–CPSF6-358 samples from fractions P1 (left) and P2 (right), as shown in panel B. Scale bars, 100 nm.

FIG 2

Characterization of CPSF6-358 oligomerization states. (A) SEC-MALS analysis of His₆-albumin–CPSF6-358 samples from P1 (black) and P2 (red) samples, shown in Fig. 1; the estimated molecular mass of the monomeric form of the protein should be 110 kDa. (B) Superdex 200 gel filtration of CPSF6-358 after TEV cleavage of the His₆-albumin tag of P2 (top) with an EM image of the purified CPSF6-358 fraction from the position of the peak indicated by the arrow (inset) and SDS-PAGE of the corresponding peaks, stained with Coomassie blue (bottom). (C) Analytical ultracentrifugation analysis of His₆-albumin–CPSF6-358 from P1 (blue), P2 (black), and CPSF6-358 (red) at 1.0 mg/ml. The expected oligomeric state for each peak is indicated.

FIG 3

CPSF6-358 binds and disrupts WT CA tubular assemblies. (A) SDS-PAGE of WT and N74D CA assemblies, following incubation with His₆-albumin–CPSF6-358, from P1 or P2 and centrifugation. The gel was Coomassie blue stained, with supernatant (s) and pellet (p) samples indicated. (B) SDS-PAGE of WT and N74D CA assemblies following incubation with untagged CPSF6-358 and centrifugation. (C to H) Representative negative-stain EM micrographs of the samples in panel A. (C to E) WT CA tubular assemblies alone (C) or with 30 μM P1 (D) or 30 μM P2 (E) His₆-albumin–CPSF6-358. (F to H) CA N74D alone (F) or with 30 μM P1 (G) or 30 μM P2 (H) His₆-albumin–CPSF6-358. The arrows indicate the capsid fragments. (I to L) Representative negative-stain EM micrographs of the samples in panel B. Shown are WT CA tubular assemblies alone (I) or with 30 μM CPSF6-358 (J) and CA N74D tubular assemblies alone (K) or with 30 μM CPSF6-358 (L). Scale bars, 100 nm. (M) Dose-dependent effect of CPSF6-358 on CA tubes. Shown is binding of P1 (blue), P2 (black), and CPSF6-358 (red) to assembled WT CA tubes (left). The effects of P1 (blue), P2 (black), and CPSF6-358 (red) binding on the average length of tubes (middle) and on the number of remaining initial tubular assemblies (right) were measured. The error bars indicate the standard deviation of the values.

FIG 4

Binding of CPSF6-358 with 14C/45C/W184A/M185A hexamer. (A to C) Gel filtration (Superdex 200) profile of CA hexamer with His₆-albumin–CPSF6-358 from P1 (A) or P2 (B) or with untagged CPSF6-358 (C). Red, CA hexamer alone; blue, CPSF6-358 proteins alone; black, mixtures. (D) SDS-PAGE analysis of fractions in panels A to C.

FIG 5

Generation of fluorescently labeled HIV-1. (A) Schematic design of the Vpr-mRuby3-IN construct used to label HIV-1 particles in trans. (B) Specific infectivity (luciferase per nanogram of p24) was measured for D116N HIV-1 complemented in trans with no plasmid, Vpr-RT, Vpr-IN, or Vpr-mRuby3-IN. (C) TIRF image of WT HIV-1 labeled with Vpr-mRuby3-IN. (D) Confocal image of HeLa cells synchronously infected with WT HIV-1 labeled with Vpr-mRuby3-IN and 5-ethynl uridine and fixed 30 min postinfection.

FIG 6

WT HIV-1 infection induces formation of CPSF6-358 higher-order complexes in HeLa cells. (A) Confocal images of HeLa cells stably expressing CPSF6-358–eGFP before or 30 min after infection with WT HIV-1 or N74D HIV-1. (B) CPSF6-358–eGFP puncta and mRuby-IN particles were quantified per cell (n ≥ 25 z-stacks) at 30 min postinfection with WT HIV-1 in the presence or absence of 10 μM PF-74, N74D HIV-1, or A77V HIV-1. The asterisks denote comparisons with P values of <0.05. (C) HeLa cells stably expressing CPSF6-358–eGFP were treated (open symbols) or not (solid symbols) with 2 μM CsA and synchronously infected with WT HIV-1 or N74D HIV-1. The number of CPSF6-358–eGFP puncta per field of view was determined. The error bars represent standard error of the mean (SEM). *, P < 0.05; **, P < 0.005; ***, P < 0.001.

FIG 7

Dynamic interactions occur between CPSF6-358 and WT HIV-1 particles. (A) Images were obtained by live-cell frustrated TIRF imaging 10 min after synchronized infection with WT HIV-1 of HeLa cells stably expressing CPSF6-358–eGFP. The arrowheads indicate initial colocalization of CPSF6-358–eGFP (green) with mRuby3-IN (red) and then separation approximately 3 min later. (B) eGFP and mRuby3 colocalized particles were quantified at 10, 30, and 60 min postinfection. The error bars represent SEM. ****, P < 0.0001.

FIG 8

Capsid permeabilization of WT HIV-1 occurs more quickly in HeLa cells expressing CPSF6-358–eGFP. HeLa cells and HeLa cells expressing CPSF6-358–eGFP were infected with WT HIV-1 (A) or N74D HIV-1 (B) and stained for viral RNA at different times. The error bars represent SEM of two (WT) or one (N74D) independent experiment. *, P < 0.05; ***, P < 0.001.