title: Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.
abstract: 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.
authors: Toxicology division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow, India.
authors: Reshu
authors: R
authors: Saxena
authors: Toxicology division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow, India.
authors: Sudipti
authors: S
authors: Gupta
authors: Electron Microscopy Lab, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow, India.
authors: Kavita
authors: K
authors: Singh
authors: Electron Microscopy Lab, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow, India.
authors: Kalyan
authors: K
authors: Mitra
authors: Department of Medicine, King George's Medical University, Chowk, Lucknow, India.
authors: Anil Kumar
authors: AK
authors: Tripathi
authors: Toxicology division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow, India.
authors: Raj Kamal
authors: RK
authors: Tripathi
mesh_terms: D015967:Gene Expression Regulation, Viral; D014644:Genetic Variation; D015658:HIV Infections / Q000235:genetics* / Q000473:pathology / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity; D006790:Host-Parasite Interactions / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D040901:Proteomics; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D054311:nef Gene Products, Human Immunodeficiency Virus