Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Abstract

BackgroundChanges in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.MethodsRT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.ResultsHere, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.ConclusionsOur study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-017-0126-0) contains supplementary material, which is available to authorized users.

Combination of chemotherapy and PD-1 blockade induces T cell responses to tumor non-mutated neoantigens

“…SILAC labeling of primary NSCLC cells and sorting. Primary NSCLC cells were grown in either SILAC heavy ( 13 C 6 15 N4-arginine and 13 C 6 -lysine) or SILAC light ( 12 C 6 14 N 4 -arginine and 12 C 6 -lysine) conditions for eight passages before the first experiment 55 . This period lasted about 3 weeks, until the SILAC heavy cells labeling was complete (SILAC Protein Quantitation Kit, cod.…”

“…NanoLC and MS analyses. Protein digestion, peptide purification, nanoLC analysis, and MS analysis were performed as previously reported 55 . Equal protein amounts (100 μg) of whole-cell extracts from CDDP-treated or -untreated NSCLC cell lines were mixed and separated on 4-12% gradient gels by SDS-PAGE (NuPAGE bis-tris gel cod.…”

“…Peptides were re-suspended with 40 μL of 2.5% ACN/0.1% TFA, desalted and filtered through a C18 microcolumn ZipTip (cod. ZTC185096, Millipore Merck, Germany), and eluted from the C18 bed using 10 μL of 80% ACN/0.1% TFA 55 . The organic component was once again removed by evaporation in a vacuum centrifuge and peptides were re-suspended in a suitable nanoLC injection volume (typically 3-10 μL) of 2.5% ACN/0.1% TFA.…”

“…SILAC-based MS was used to identify changes in the proteome of primary NSCLC cells, as previously described 55 (Fig. 1).…”

Role of Divalent Cations in HIV-1 Replication and Pathogenicity

“…Iron may also decrease HIV-1 by restricting the ability of HIV-1 Tat to bind to the TAR region of the HIV-1 LTR promoter [238]. Iron overload also suppressed HIV-1 replication by decreasing Rev cofactor eIF5α [239].…”