Fig 1.
Mature p6 is detectable only in released virus particles.
HEK293T cells were transfected with the env-deleted HIV-1 expression plasmid pNLenv1 (Δenv). Cell lysates and VLP fractions were analyzed by Western blotting using a p6-reactive (A) or a CA-reactive (B) antiserum. (C) Schematic depiction of Gag processing products observable in A and B. (D) 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.
Fig 2.
p6 is expressed as a YFP-fusion, but is highly turned over when expressed as an autonomous protein.
(A) Schematic depiction of the CMV-driven expression plasmids. For ectopic expression of p6, a codon-optimized p6 gene was fused to YFP and cloned into the vector pcDNA (p6-YFP). By insertion of a stop codon (*) after the p6 ORF, translation of YFP-fusion part was abrogated. Additional vectors were established with an AU1 tag or a SIINFEKL (SL)-epitope at the C-terminus of the p6 ORF. For control, the Vpu-AU1 construct encodes a Vpu protein harboring an AU1 tag. (B) HeLa cells were transfected with plasmids indicated and expression of p6-YFP fusion was detected with antibodies specific for GFP or p6. Staining for the ribosomal P-antigen (ribP0) served as loading control. (C) Cytosolic extracts from HeLa cells transfected with indicated constructs were analyzed by Western blotting using antibodies specific for AU1 or p6. 20 ng of sp6 served as control for the anti-p6 staining. (D) HeLa-Kb cells were transfected with p6-expression constructs, coding for p6 or p6-SL and as an empty vector control pcDNA. H2-Kb-SL complexes presented on the cell surface were quantified by flow cytometry using the mAb 25D1.16-APC. Inset: the total amount of H2-Kb complexes was determined by the mAb B8-24-3. Quantification of five independent experiments; Bars represent mean values ± SD.
Fig 3.
Cytoplasmic S10 from HeLa cells contains an enzymatic activity that degrades sp6 and vp6.
(A) 100 ng sp6 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) 100 ng sp6 were incubated with 5 μg S10 extract for the times indicated at 37°C. (C) Amounts of p6 were quantified for four independently performed experiments. Values represent the arithmetic mean ± SD. (D) 10 ng sp6BY were incubated with 5 μg S10 extract for 30 min at 37°C. sp6BY was detected by measurement of fluorescence excitation. (E) 10 ng sp6BY 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. (F) VLPs produced in HEK293T cells transfected with the subgenomic HIV-1 expression plasmid pΔR [11] 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. (G) VLPs were produced and treated as described in (F) and analyzed for Vpr content.
Fig 4.
The 51 aa EIAV p9 protein and HIV-2 or SIV p6 are not degraded in S10.
(A) 400 ng of HIV-2 or SIV sp6, or 100 ng of HIV-1 sp6, or EIAV sp9 were incubated with 5 μg S10 extract for the indicated times at 37°C, and remaining sp6 or sp9 was detected by Western blot. (B) Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. (C) 10 ng sp6BY were incubated with 5 μg S10 and increasing concentrations of HIV-1, HIV-2 or SIV sp6 or EIAV sp9 for 30 min at 37°C. sp6BY was detected by measurement of fluorescence excitation. (D) Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. (E) 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. (F) Band intensities were quantified for three independently performed experiments. Values represent the arithmetic mean ± SD. (G) 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 EIAVWyoming [29].
Fig 5.
Inhibitors of the metalloprotease IDE block the in vitro degradation of sp6.
(A) 100 ng sp6 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 sp6BY were incubated with 5 μg S10 extract and increasing concentrations of insulin (B) or 6bK (C) for 30 min at 37°C. sp6BY was detected by measurement of fluorescence excitation. Values represent the arithmetic mean ± SD of at least three independent experiments.
Fig 6.
IDE is sufficient and required for the in vitro degradation of sp6 and vp6.
(A) 10 ng sp6BY were incubated with 5 μg S10 extract from HeLa cells, either untransfected or transfected with control or IDE-specific siRNA, for 0, 10, 30 or 90 min at 37°C. (B) sp6BY was detected by fluorescence emission and quantified. (C) VLP lysates from HEK293T cells were incubated with S10 from siRNA treated cells described in A. Viral proteins were detected as described in Fig 1. (D) Western blot analyses of the S10 extract used in A and C. The membrane was stained with antibodies specific for IDE, HSP70, and β-actin. (E) 10 ng rIDE were incubated with 10 ng sp6BY and either TPEN (1 mM), NEM (1 mM), insulin (100 μg/ml) or buffer for 30 min at 37°C. (F) VLP lysates were incubated with 10 ng rIDE for 30 min.
Fig 7.
sp6 is an up to 100-fold better IDE-substrate than insulin.
(A) 10 ng of sp6BY or insulin-FITC were incubated with indicated amounts of rIDE for 30 min at 37°C. Remaining amounts of sp6BY and insulin-FITC were detected by measurement of fluorescence excitation. (B) Results of four independently performed experiments. Values represent the arithmetic mean ± SD. (C) Increasing concentrations of sp6BY were incubated with rIDE for 10 min at 37°C. The velocities were calculated from the degradation of sp6BY 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. (D) Comparison of KM and vmax values for sp6BY and IDE as determined in (C) to those of IDE and insulin or Aβ as reported [47].
Fig 8.
Generation of stable p6 mutants.
(A) 100 ng sp6 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) p6 mutants that encode multiple PTAPPA- or LTAPPA-motifs were generated. The introduced amino acids are underlined. (C) 10 ng of sp6BY were incubated with 250 μg/ml sp6 wt, 2xPTAPPA, 3xPTAPPA or only buffer and 5 μg S10 for 30 min at 37°C. sp6BY was detected by fluorescence emission and quantified. Values represent the arithmetic mean ± SD of four independent experiments.
Fig 9.
Multiplication of PTAPPA-motifs stabilizes p6.
20 ng of sp6 or 30 ng of vp6 were incubated either with 5 μg S10 extract (A/B/C) or 2 ng of rIDE (D) for up to 60 min. Degradation efficiency was quantified via densitometric analyses of Western blots. Values represent the arithmetic mean ± SD of at least 3 independent experiments for each setting.
Fig 10.
The IDE-p6-interaction has no effect on Gag-processing and virus release.
(A) HEK293T cells were transfected with pΔR plasmids encoding for either wt 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 wt, only the apparent molecular weight of p6 and the NCp6 processing intermediate increased by PTAPPA multiplication. (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. (C) 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 wt were set to 1. (D) HAP1 wt cells and HAP1 IDE knock out cells were infected with VSV-G-pseudotyped wt 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 (E) and Vpr (F) were quantified and normalized for p24 signals. Bars represent mean values of three independent experiments ± SD. (G) HeLa TZM-bl wt and IDE KO cells were transiently transfected with pNLenv1 and virus and cell fractions were analyzed by Western blot. (H) Band intensities of virus-associated p6 were quantified as described in (E).
Fig 11.
The stability of p6 correlates inversely with the replication capacity of HIV-1 and sensitivity to insulin in X4-tropic replication.
(A) A representative replication profile of HIV-1NL4-3 variants is shown for PHA-IL2-stimulated PBMCs, infected with wt, 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-1NL4-3 wt, 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-1NL4-3 wt in each experiment was set to 100%. Error bars, ± SD (Inset). (B) Replication capacity of X4-tropic HIV-1NL4-3 wt 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-1NL4-3 wt from each experiment was set to 100%. Error bars, ± SD. (C) Replication capacity of R5-tropic HIV-1NL4-3 wt 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-1NL4-3 wt from each experiment was set to 100%. Error bars, ± SD. (D) PHA-IL2-stimulated PBMCs were infected with HIV-1NL4-3 wt 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 (E) or R5 tropic (F) viruses. (G) Cell viability was assessed by water-soluble tetrazolium salt assay on the last day of replication study.