Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

<div><p>There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.</p></div

Cytoplasmic S10 from HeLa cells contains an enzymatic activity that degrades <i>s</i>p6 and <i>v</i>p6.

<p><b>(A)</b> 100 ng <i>s</i>p6 were incubated with 5 μg S10 extract from HeLa cells for 30 min at 37°C. In one reaction, S10 extract was heat-inactivated (95°C, 5 min) prior to incubation (*). <b>(B)</b> 100 ng <i>s</i>p6 were incubated with 5 μg S10 extract for the times indicated at 37°C. <b>(C)</b> Amounts of p6 were quantified for four independently performed experiments. Values represent the arithmetic mean ± SD. <b>(D)</b> 10 ng <i>s</i>p6BY were incubated with 5 μg S10 extract for 30 min at 37°C. <i>s</i>p6BY was detected by measurement of fluorescence excitation. <b>(E)</b> 10 ng <i>s</i>p6BY were incubated with 5 μg S10 extract for the times indicated. Band intensities were quantified with AIDA for seven independently performed experiments. Values represent the arithmetic mean ± SD. <b>(F)</b> VLPs produced in HEK293T cells transfected with the subgenomic HIV-1 expression plasmid pΔR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174254#pone.0174254.ref011" target="_blank">11</a>] were isolated, lysed with 0.5% Triton X-100 and incubated with 5 μg S10 extract for 30 min at 37°C. (*) S10 extract, or VLP lysate, was heat-inactivated for 5 min at 95°C prior to incubation. Samples were analyzed by Western blotting. <b>(G)</b> VLPs were produced and treated as described in (F) and analyzed for Vpr content.</p

Inhibitors of the metalloprotease IDE block the <i>in vitro</i> degradation of <i>s</i>p6.

<p><b>(A)</b> 100 ng <i>s</i>p6 were incubated without or with indicated inhibitors and 5 μg S10 extract for 30 min at 37°C. Remaining p6 was detected by Western blot. 10 ng of <i>s</i>p6BY were incubated with 5 μg S10 extract and increasing concentrations of insulin <b>(B)</b> or 6bK <b>(C)</b> for 30 min at 37°C. <i>s</i>p6BY was detected by measurement of fluorescence excitation. Values represent the arithmetic mean ± SD of at least three independent experiments.</p

Multiplication of PTAPPA-motifs stabilizes p6.

<p>20 ng of <i>s</i>p6 or 30 ng of <i>v</i>p6 were incubated either with 5 μg S10 extract <b>(A/B/C)</b> or 2 ng of rIDE <b>(D)</b> for up to 60 min. Degradation efficiency was quantified <i>via</i> densitometric analyses of Western blots. Values represent the arithmetic mean ± SD of at least 3 independent experiments for each setting.</p

The 51 aa EIAV p9 protein and HIV-2 or SIV p6 are not degraded in S10.

<p><b>(A)</b> 400 ng of HIV-2 or SIV <i>s</i>p6, or 100 ng of HIV-1 <i>s</i>p6, or EIAV <i>s</i>p9 were incubated with 5 μg S10 extract for the indicated times at 37°C, and remaining <i>s</i>p6 or <i>s</i>p9 was detected by Western blot. <b>(B)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(C)</b> 10 ng <i>s</i>p6BY were incubated with 5 μg S10 and increasing concentrations of HIV-1, HIV-2 or SIV <i>s</i>p6 or EIAV <i>s</i>p9 for 30 min at 37°C. <i>s</i>p6BY was detected by measurement of fluorescence excitation. <b>(D)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(E)</b> Virions produced in HEK293T cells transfected with expression plasmids pNLgp2/Udel-1 (HIV-2) or pSIV3+ (SIV) were isolated, lysed with 0.5% Triton X-100 and incubated with 5 μg S10 extract or 10 ng rIDE for 30 min at 37°C. <b>(F)</b> Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. <b>(G)</b> Sequence alignment of p6 peptides from HIV-1, HIV-2, SIV and the EIAV p9 peptide. The sequence of HIV-2 p6 originates from the isolate ROD10, SIV p6 from SIVmac239, and EIAV p9 from the isolate EIAV_Wyoming [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174254#pone.0174254.ref029" target="_blank">29</a>].</p

The stability of p6 correlates inversely with the replication capacity of HIV-1 and sensitivity to insulin in X4-tropic replication.

<p><b>(A</b>) A representative replication profile of HIV-1_NL4-3 variants is shown for PHA-IL2-stimulated PBMCs, infected with <i>wt</i>, 2xPTAPPA (2x), 3xPTAPPA (3x) (30 pg p24, MOI 10⁻⁴) or mock infected, and replication was assessed by quantification of the virus-associated reverse transcriptase (RT) activity contained in cell culture supernatant collected on the indicated days post infection (dpi). The replication capacity of X4-tropic HIV-1_NL4-3 <i>wt</i>, 2xPTAPPA or 3xPTAPPA following infection of PHA-IL2-stimulated PBMCs from 6 different donors was assessed by calculating the area under the curve (AUC) from each individual replication profile. The replication capacity of HIV-1_NL4-3 <i>wt</i> in each experiment was set to 100%. Error bars, ± SD (Inset). (<b>B</b>) Replication capacity of X4-tropic HIV-1_NL4-3 <i>wt</i> or 3xPTAPPA with or without permanent treatment with 50 μg/ml insulin following infection of PHA-IL2-stimulated PBMCs from 3 different donors. The replication capacity of HIV-1_NL4-3 <i>wt</i> from each experiment was set to 100%. Error bars, ± SD. (<b>C</b>) Replication capacity of R5-tropic HIV-1_NL4-3 <i>wt</i> or 3xPTAPPA with or without permanent treatment with 50 μg/ml insulin following infection of PHA-IL2-stimulated PBMCs from 3 different donors. The replication capacity of HIV-1_NL4-3 <i>wt</i> from each experiment was set to 100%. Error bars, ± SD. <b>(D)</b> PHA-IL2-stimulated PBMCs were infected with HIV-1_NL4-3 <i>wt</i> or 3xPTAPPA and permanently treated with 10 μM 6bK, or were left untreated. Replication capacities were determined as described in (B) for PHA/IL-2-stimulated PBMCs from 3 different donors following infection with X4 <b>(E)</b> or R5 tropic <b>(F)</b> viruses. <b>(G)</b> Cell viability was assessed by water-soluble tetrazolium salt assay on the last day of replication study.</p

Generation of stable p6 mutants.

<p><b>(A)</b> 100 ng <i>s</i>p6 were incubated with 10 ng rIDE for 1, 5, 10, 30 or 60 min at 37°C. Reactions were stopped by adjusting the samples to 0.3% (w/v) TFE and subsequently analyzed by mass spectrometry. Arrows above the primary sequence represent the detected cleavage sites, and initial and secondary cleavage sites are indicated as big or small arrows, respectively. Red font indicates positively charged, and blue negatively charged residues. Previously identified α-helices and binding motifs are depicted below the primary sequence. <b>(B)</b> p6 mutants that encode multiple PTAPPA- or LTAPPA-motifs were generated. The introduced amino acids are underlined. <b>(C)</b> 10 ng of <i>s</i>p6BY were incubated with 250 μg/ml <i>s</i>p6 <i>wt</i>, 2xPTAPPA, 3xPTAPPA or only buffer and 5 μg S10 for 30 min at 37°C. <i>s</i>p6BY was detected by fluorescence emission and quantified. Values represent the arithmetic mean ± SD of four independent experiments.</p

The IDE-p6-interaction has no effect on Gag-processing and virus release.

<p><b>(A)</b> HEK293T cells were transfected with pΔR plasmids encoding for either <i>wt</i> Gag, or the p6 mutants 2xPTAPPA or 3xPTAPPA. Cells were lysed and VLPs were purified and subsequently analyzed by Western blot. Noteworthy, Gag processing and virus release of 2x and 3x PTAPPA mutants were comparable to that of the <i>wt</i>, only the apparent molecular weight of p6 and the NCp6 processing intermediate increased by PTAPPA multiplication. <b>(B)</b> The rate of Gag processing was estimated by calculating the ratio of p24 vs. Pr55 detected in released VLPs. Bars represent mean values of three independent experiments ± SD. <b>(C)</b> Efficiency of virus release was calculated as the ratio of Gag (Pr55 and p24) present in the virus pellet relative to the total amount of Gag detected in cells and released VLPs. Bars represent mean values of three independent experiments ± SD. Both Gag processing and virus release for the <i>wt</i> were set to 1. <b>(D)</b> HAP1 <i>wt</i> cells and HAP1 IDE knock out cells were infected with VSV-G-pseudotyped <i>wt</i> HIV-1 particles. 2 days post-infection, cell and virus-fractions were harvested and analyzed by Western blot for viral proteins. Band intensities of virus-associated p6 <b>(E)</b> and Vpr <b>(F)</b> were quantified and normalized for p24 signals. Bars represent mean values of three independent experiments ± SD. <b>(G)</b> HeLa TZM-bl <i>wt</i> and IDE KO cells were transiently transfected with pNLenv1 and virus and cell fractions were analyzed by Western blot. <b>(H)</b> Band intensities of virus-associated p6 were quantified as described in (E).</p

<i>s</i>p6 is an up to 100-fold better IDE-substrate than insulin.

<p><b>(A)</b> 10 ng of <i>s</i>p6BY or insulin-FITC were incubated with indicated amounts of rIDE for 30 min at 37°C. Remaining amounts of <i>s</i>p6BY and insulin-FITC were detected by measurement of fluorescence excitation. <b>(B)</b> Results of four independently performed experiments. Values represent the arithmetic mean ± SD. <b>(C)</b> Increasing concentrations of <i>s</i>p6BY were incubated with rIDE for 10 min at 37°C. The velocities were calculated from the degradation of <i>s</i>p6BY and normalized for the amount of rIDE. Data points represent values from three independent experiments in a double-reciprocal Lineweaver-Burk plot. The inset shows a magnification of the intersections of the regression lines with the axes. <b>(D)</b> Comparison of K_M and v_max values for <i>s</i>p6BY and IDE as determined in (C) to those of IDE and insulin or Aβ as reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174254#pone.0174254.ref047" target="_blank">47</a>].</p

Mature p6 is detectable only in released virus particles.

<p>HEK293T cells were transfected with the <i>env</i>-deleted HIV-1 expression plasmid pNL<i>env</i>1 (Δenv). Cell lysates and VLP fractions were analyzed by Western blotting using a p6-reactive (<b>A</b>) or a CA-reactive (<b>B</b>) antiserum. (<b>C</b>) Schematic depiction of Gag processing products observable in A and B. (<b>D</b>) Quantitative analysis of mature p6 versus total amount of p6-containing Gag proteins for cell and VLP fractions. Values represent the arithmetic mean of 3 independently performed experiments ± SD.</p