Non-Metabolic Role of PKM2 in Regulation of the HIV-1 LTR
Authors: Rafal Kaminski
Satish L Deshmane
Satarupa Sen
Prasun K Datta
Shohreh Amini
Journal: Journal of Cellular Physiology (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: cellular proteins; transcription factors; HIV-1; replication; monocytes
Affiliations: Department of Neuroscience, Comprehensive NeuroAIDS Center, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania., Department of Biology, College of Science and Technology, Philadelphia, Pennsylvania.

Abstract:  Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPβ, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning -120 bp to -80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517-525, 2017. © 2016 Wiley Periodicals, Inc. 

Retracted: No

PubPeer comments:
 In Figure 4, the Lane M, DNA size ladder for both A and B appears unexpectedly similar. When the 2 lanes are overlaid ( stretching B), the 2 images cancel out. Could the authors comment? *** Overlay ![file] *** Original ![file] Fig. 4. (A) The interaction of PKM2 and p65 in vivo with HIV-1 LTR in U1 cells induced with PMA was analyzed using ChIP analysis. DNA isolated from chromatin immunoprecipitated with Lane 1: normal rabbit IgG (negative control, IgG); Lane 2: p65 antibodies (p65), and lane 3: PKM2 antibodies (PKM2) was amplified by PCR using primers described in the materials and methods. Lane 4: Total chromatin used as the input (input) and amplified by PCR. Lane: M: DNA size ladder (B). The interaction of PKM2 and p65 in vivo with HIV-1 LTR in TZM-bl cells induced with PMA was analyzed using ChIP analysis. DNA isolated from chromatin immunoprecipitated with Lane 1: normal rabbit IgG (negative control, IgG); Lane 2: p65 antibodies (p65) and lane 3: PKM2 antibodies (PKM2) was amplified by PCR using primers described in the materials and methods. Lane 4: Total chromatin used as the input (lane: input) and amplified by PCR. Lane: M: DNA size ladder. ,