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. 2015 Oct;89(19):9765-80.
doi: 10.1128/JVI.01522-15. Epub 2015 Jul 15.

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: A Role for Integrase in Maturation

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Free PMC article

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: A Role for Integrase in Maturation

Juan Fontana et al. J Virol. .
Free PMC article

Abstract

During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼ 12% to ∼ 53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.

Importance: Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.

Figures

FIG 1
FIG 1
Chemical structures of BI-D and BIB-2.
FIG 2
FIG 2
Inhibition of virion maturation underlies ALLINI potency. (A) (Left) Quantitation of core morphotype frequencies (average ± SD for two independent experiments) for sets of 100 counted HIV-Luc particles; (right) representative TEM images of mature, eccentric, and immature virions produced in these experiments. (B) Quantitation of late reverse transcription (LRT) product formation from infections initiated with samples shown in panel A. (C) Antiviral activity as assessed by bulk luciferase output. Error bars in panels B and C represent the variations obtained from three independent experiments.
FIG 3
FIG 3
Classification of HIV-1 particles observed by cryo-ET. (A to D) Tomographic central slices of representative HIV-1 virions classified according to core morphology (conical or nonconical) and the presence or absence of an eccentric condensate (white arrows). Eccentric condensates appear denser than the material lying between the core and the envelope in particles that lack an eccentric condensate. The cores of virions that have an eccentric condensate are relatively empty (e.g., panel B). Bar, 50 nm. (E) Percentages of HIV-1 virions classified according to their core morphology (conical, nonconical, or no core) and presence or absence of an eccentric condensate. The majority species for each sample is indicated by values in bold. According to the chi-square test, significant differences with P values of <0.00001 were found for comparisons between WT and BIB-2 and between WT and the V165A mutant. The difference between WT and BI-D had a P value of 0.013, whereas the differences between the V165A mutant and BI-D or BIB-II were not significant, with P values of >0.26. The chi-square test was used to test for equal distribution of compartmentalized data but should not be used if more of 20% of the bins contain fewer than 5 counts. Therefore, immature virions were not taken into account and virions with no cores were considered a single category, independent of the presence or absence of an eccentric condensate.
FIG 4
FIG 4
Examples of relatively rare morphotypes observed in populations of WT virions. (A) Central tomographic slice of a WT particle without a core or eccentric condensate; (B) central tomographic slice (left) and corresponding tomo-bubblegram slice (right) of a WT particle containing an eccentric condensate (marked with an arrow in the left panel). The core is a conical one sampled in an oblique section. While the core is not entirely empty, it lacks sufficient internal density to suggest that it contains a vRNP. This interpretation is confirmed by the bubbling being confined to the eccentric condensate. Bar, 50 nm.
FIG 5
FIG 5
Tomo-bubblegrams of HIV-1 particles. (A to X) Each pair of panels shows a central section from an initial tomogram (left) and the corresponding section from the tomo-bubblegram (right). The types of virions analyzed are indicated on the left of each block. BIB-2 (10×) and BI-D (10× EC50 for panels G, I, and L; 100× EC50 for panels H, J, and K) were used at full inhibitory concentrations. Particles with conical cores are framed in red, and those with nonconical cores are framed in blue; particles lacking eccentric condensates are framed with continuous lines, and particles that have eccentric condensates are framed with dashed lines. Bar, 50 nm. Full tomograms of the virions in panels B (Movie S1), I (Movie S2), R (Movie S3), and X (Movie S4) are shown in the supplemental material.
FIG 6
FIG 6
Tomo-bubblegrams of immature HIV-1 particles. (Ai to Avi) Central tomographic sections (left) and corresponding tomo-bubblegram sections (right). Full tomograms of the virions in panels Aii (Movie S5), Aiii (Movie S6), and Avi (Movie S7) are shown in movies in the supplemental material. The diagrams on the left side of the figure illustrate the composition of each sample. PR particles contain the Gag and Gag-Pol polyproteins that include NC and incorporate vRNA. The virions are immature due to the inactivating D25A mutation in the PR active site. Gag/ψ-minus particles are also immature, and additionally their RNA lacks the ψ region and therefore vRNA is not incorporated into particles. The Gag-LeuZip particles replace NC with a leucine zipper; these particles lack NC and vRNA. Bar, 50 nm. (B) Total virion RNA from the particles described for panel A. RNA concentration was normalized to the Gag signal obtained from anti-p24 Western blots of multiple input volumes. Shown are the averages (±SD) of total RNA (in nanograms) standardized by Gag signal for four independent experiments, reported as a percentage of PR. (C) RNA from panel B was utilized to determine the copy number of vRNA. Values, reported as a percentage of PR, are the averages (±SD) for four independent experiments.
FIG 7
FIG 7
Locations of bubbles in PR virions. (A) Cryo-ET central slice of an immature PR particle. (B and C) Tomo-bubblegram section corresponding to panel A. In panel C, bubbles are marked with red circles. (D) Surface rendering of a cutaway slice of the particle shown in panels A to C. Most of the bubbles (white) are located between the CA layer (green) and the NC-vRNA layer (yellow). The viral envelope is in cyan, and the internal contents are in red. Bar, 50 nm. (E) Radial density profile of subtomogram averaged Gag from immature virions (from reference 25). (F) Histogram of the distance from the center of the bubbles to the center of the viral envelope (membrane + MA).
FIG 8
FIG 8
Bubblegram imaging of purified NC protein. (A to E) Cryo-EM dose series of five NC aggregates of different sizes. The images in which bubbles first appear are labeled with a white asterisk in each case (although NC is formally soluble, small aggregates of suitable size were consistently found on EM grids). Bar, 50 nm. (F) Plot of the diameters of NC aggregates versus the cumulative electron dose at which they started bubbling. Points corresponding to the aggregates from panels A to E are shown in red. While the data exhibit some stochastic variability in bubbling threshold, there is a clear trend toward larger aggregates requiring less electron radiation to initiate bubbling. A linear fit is shown for reference (red line).
FIG 9
FIG 9
Viral RNA requirement for eccentric condensate formation. (A) Total RNA (in nanograms) of concentrated virion preparations. RNA concentration was standardized by the p24 signal obtained from Western blots of multiple input volumes. Shown are averages (±SD) from four independent experiments reported as percentages of untreated HIV-Luc virions. (B) Average (±SD) copies of vRNA per ng total RNA (n = 4) determined for the samples described for panel A. (C) (Left) Core morphology frequencies in sets of 100 counted virions (average ± SD) from two independent isolates of particles prepared as described for panel A. **, P < 0.01; n.s., not significant (P = 0.37). (Right) Representative images of the mature, eccentric, dual electron density (ED), empty, and immature virions observed in these experiments.
FIG 10
FIG 10
IN in trans stimulates WT core formation in ΔIN virions. (A) Infectivity of HIV-Luc.ΔIN transcomplemented with Vpr-INWT or Vpr-INH171T made in the presence of BI-D or DMSO. Values were normalized to control HIV-Luc virus constructed in the presence of DMSO. (B) (Upper) Quantitation of core morphology frequencies of 100 counted virions (the ΔIN mutation yields more immature particles than does other class II [e.g., missense] mutations [18, 46]). Shown are averages ± SD for two independent experiments for the transcomplemented preparations described for panel A. (Lower) Representative images of the mature, eccentric, and immature virions observed within experiments.
FIG 11
FIG 11
Model for the role of IN in coordinating HIV-1 capsid assembly and vRNP incorporation into the mature core. Maturation starts with activation of the PR and dissection of the Gag and Gag-Pol polyproteins and release of the vRNP or NC from the Gag shell. The key element of this model is that it envisages a molecular complex containing one or several copies of Gag-Pol that initiates both assembly of the capsid by an outgrowth of CA subunits and packaging of the vRNA through binding to IN or to RT as conformationally influenced by IN. Many details remain to be clarified, including, for instance, whether other components are involved in the putative complex, whether the initiation process starts with uncleaved Gag-Pol or whether the polyproteins have been proteolytically processed, with the cleavage products remaining together, and whether capsid assembly begins at the wide or narrow end. We favor the wide end because this narrows down to give a capsid of about the right length, which CA has been shown to be capable of in the absence of a membrane (71). The cartoon shows RT and IN associated with the eccentric condensate for which there is currently no evidence, although they do remain, mostly or totally, inside the wild-type capsid (66). A variant of this model in the Rous sarcoma virus system envisages participation of the cytoplasmic domain of Env (72). The virion and core also contain other components (e.g., Vpr in the core and some host proteins somewhere in the virion), which are not shown.

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