Western blot confirmation of LC-MS/MS identified proteins.

<p>Western blotting of representative proteins revealed similar quantitative profiles as suggested by 2DGE analysis. Contrasting effect of Nef variants observed upon expression of the six proteins- Cyclophilin A, EIF5A-1, Rho GDI, VDAC1, OTUB1, and ENO1. alpha tubulin was used as a loading control. Data are presented as the mean ± SD of three independent experiment.</p

Western blot confirmation of LC-MS/MS identified proteins.

<p>Western blotting of representative proteins revealed similar quantitative profiles as suggested by 2DGE analysis. Contrasting effect of Nef variants observed upon expression of the six proteins- Cyclophilin A, EIF5A-1, Rho GDI, VDAC1, OTUB1, and ENO1. alpha tubulin was used as a loading control. Data are presented as the mean ± SD of three independent experiment.</p

Immunofluorescence staining of SupT1 cells showing comparative expression of downregulated proteins.

<p>Immunofluorescence study to compare cell surface expression of six proteins in SupT1 cells (1 μm section) was captured by Confocal microscopy at 40 X and 63 X magnification. Figure shows immuostained SupT1 cells for expression of Vector/Nef (green), respective proteins (red) with their nucleus stained with DAPI (blue). Fluorescence intensity was measured for 5–8 Nef/vector transfected cells from 6 different fields of each sample and mean was calculated. Data is presented as values from two different experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122994#pone.0122994.g005" target="_blank">Fig 5</a> displays ENO1 and VDAC1 (A-C) and EIF5A, OTUB1, CYPA and RhoGDI (D-H).</p

Protein-protein interactions of identified Nef downregulated proteins.

<p>Predicted network map for Cyclophilin A, EIF5A-1, Rho GDI, VDAC1, OTUB1, and ENO1 with their top partners and association with HIV-1 Nef was created in STRING. Proteins in boxes were underexpressed proteins identified in our work. Rest of proteins were host proteins from the database. Protein labeled in Yellow is Nef, with known interaction with Cyclophilin A, RAC1, CDC42 and BCL2L1 (connected with red line) as given in HIV-1, Human Protein Interaction Database.</p

Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

<div><p>Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 _{(55AAAAAAA61)} and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.</p></div

Protein Identification Summary.

<p>List of proteins downregulated by Nef in 2D gels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122994#pone.0122994.g002" target="_blank">Fig 2(A) and 2(B)</a></p><p>a & b Theoretical mass and isoelectric point calculated by Swissprot database at <a href="http://ca.expasy.org/sprot/" target="_blank">http://ca.expasy.org/sprot/</a></p><p>c Number of peptides detected by mass spectrometry for each identified protein</p><p>d Sequence coverage (%) means the number of amino acids spanned by the assigned peptides divided by the sequence length.</p><p>e Mascot score for identified protein</p><p>f Compared with control SupT1 cells (vector transfected)</p><p>g Protein function from SWISS-PROT database</p><p>h Protein location from SWISS-PROT database</p><p>Protein Identification Summary.</p

Nef induced transcriptional alteration of the six genes measured by qPCR.

<p>Real Time RT-PCR was performed to analyse the expression of the six genes. Fold change in expression upon Nef transfection is presented in respect to control. Except VDAC1 all 5 genes were downregulated by Nef variants. The values and error bars represent average and standard deviations of three independent set of experiments. Student T test was performed to find out significant difference between control and treated conditions.</p

List of primers used for amplification in q-PCR.

<p>List of primers used for amplification in q-PCR.</p

Western blotting analysis with Nef RP01 (55AAAAAAA61) mutant.

<p>(A)Western blots showing reverse effect of Nef RP01 mutant upon downmodulation of α-enolase (ENO1) and VDAC1 as caused by Nef RP01. Alpha-tubulin was used as a loading control. (B)Graphs representing the difference in effect of Nef RP14 Nef RP01 and Nef RP01 mutant upon expression of α-enolase (ENO1) and VDAC1. Data are presented as the mean ± SD of two independent experiment.</p