MOESM2 of Infectious SIV resides in adipose tissue and induces metabolic defects in chronically infected rhesus macaques

Additional file 2. Sequence confirmation of nested PCR products, and lack of viral diversity in AT-SVF of acutely infected rhesus macaques. PCR products from SHIV Gag (A) and Env (B) 2nd round nested PCR reactions of subcutaneous and visceral AT-SVF DNA of eight infected monkeys (shown in Figure 2G) were gel-purified, sequenced, and aligned with Clustal-Omega software. Yellow-highlighted nucleotides indicate a nucleotide difference compared to other nucleotides in the alignment column (sequences include nucleotide positions A:1667 to G:2085 relative to SIVmac239 Gag, and G:7042 to C:7329 relative to HIV-1 HXB2 Env, indicated in red)

Data Mining Framework For Discovering and Clustering Phenotypes of Atypical Diabetes

Supplemental tables and figures for "Data Mining Framework For Discovering and Clustering Phenotypes of Atypical Diabetes"</p

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

Impact of insulin sensitivity and β-cell function over time on glycemic outcomes in the GRADE (Glycemia Reduction Approaches in Type 2 Diabetes: A Comparative Effectiveness) Study: differential treatment effects of dual therapy.

Objective: Compare the effects of insulin sensitivity and β-cell function over time on HbA1c and durability of glycemic control in response to dual therapy. Design and Methods: GRADE Study participants were randomized to glimepiride (n=1254), liraglutide (n=1262), or sitagliptin (n=1268) added to baseline metformin and followed for 5.0±1.3 years with HbA1c quarterly and OGTTs at baseline, 1, 3, and 5 years. We related time-varying insulin sensitivity (HOMA2-%S) and early (0-30 min) and total (0-120 min) C-peptide (CP) responses to changes in HbA1c and glycemic failure (primary: HbA1c≥7% (53 mmol/mol) and secondary outcome: HbA1c>7.5% (58 mmol/mol)) and examined differential treatment responses. Results: Higher HOMA2-%S was associated with greater initial HbA1c lowering (3 months) but not subsequent HbA1c rise. Greater CP responses were associated with a larger initial treatment response and slower subsequent HbA1c rise. Higher HOMA2-%S and CP responses were each associated with lower risk of primary and secondary outcomes. These associations differed by treatment. In the sitagliptin group, HOMA2-%S and CP responses had greater impact on initial HbA1c reduction (test heterogeneity p=0.009 HOMA2-%S; p=0.018 early CP; p=0.001 total CP) and risk of primary outcome (p=0.005 HOMA2-%S; p=0.11 early CP; p=0.025 total CP), but lower impact on HbA1c rise (p=0.175 HOMA2-%S; p=0.006 early CP; p<0.001 total CP) compared to glimepiride and liraglutide. There were no differential treatment effects on secondary outcome. Conclusion: Insulin sensitivity and β-cell function affected treatment outcomes irrespective of drug assignment, with greater impact in the sitagliptin group on initial (short-term) HbA1c response compared to glimepiride and liraglutide.</p

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

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

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

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

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

<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