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.
Highlights
Nef is a 27kDa, N-terminal myristoylated accessory protein of HIV-1, involved in disease progression and pathogenesis
The two Nef variants taken in this study were RP14 and RP01
The study adopted a gel based proteomic approach to probe changed proteins that allowed for identification of physiologically relevant targets of signal transduction pathway
Summary
Nef is a 27kDa, N-terminal myristoylated accessory protein of HIV-1, involved in disease progression and pathogenesis. It is expressed in early stage of viral lifecycle and is one of the first proteins to be detected after host cell invasion. Nef promotes the endocytosis and downregulation of cell surface proteins, including CD4 and MHC proteins [2] This action possibly impairs cytotoxic T cell function, and advances host immune evasion by virus thereby establishing state of infection [6]. Nef promotes survival of infected cells and favours viral infection and replication through interaction with cellular proteins involved in both trafficking of cell-surface receptors and signal transduction molecules [5, 7]
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