Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

Endo‐Munoz, Liliana; Warby, Tammra; Harrich, David; McMillan, Nigel A.J.

doi:10.1186/1743-422x-2-17

Cited by 56 publications

(36 citation statements)

References 41 publications

(45 reference statements)

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“…PKR has been reported to phosphorylate Tat at residues S62, T64, and S68 (24), and Tat phosphorylation at these three residues enhances Tat binding to TAR RNA (26). However, our repeated experimental results (Fig.…”

Section: Discussionmentioning

confidence: 57%

“…Tat activity is altered by cellular modification factors such as p300, p300/CBP-associating factor (PCAF), Hdm2, Hexim1, and SKIP (20)(21)(22)(23). Tat phosphorylation by PKR (24) and CDK2 (25) has also been reported, and phosphorylation of Tat by PKR at three Ser/Thr sites (S62, T64, and S68) was shown to increase Tat-Tatresponsive region (TAR) binding and enhance transcription (26). However, the detailed molecular mechanisms underlying host restriction of HIV-1 replication via p53/PKR-mediated Tat phosphorylation and inactivation after HIV-1 infection are largely unknown.…”

mentioning

confidence: 99%

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p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

Yoon

Kim

Byeon

et al. 2015

J Virol

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Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53 ؊/؊ and eIF2␣ CA cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat-Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCEHIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1. Human immunodeficiency virus type I (HIV-1) encodes a highly conserved trans-activator (Tat) protein, which is essential for viral replication and disease progression (1). Although Tat is a potent trans-activator in HIV-1 replication, HIV-1-mediated disease progression lasts for decades after primary infection (2). Host restriction mechanism...

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Section: Discussionmentioning

confidence: 57%

mentioning

confidence: 99%

p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

Yoon

Kim

Byeon

et al. 2015

J Virol

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“…Our conclusion that SG formation is PKR dependent and requires a suprathreshold concentration of cytoplasmic gRNA is compatible with evidence that cells contain endogenous PKR inhibitors including proteins with dsRBDs and inhibitory RNAs (Bannwarth and Gatignol, 2005; Brand et al, 1997; Cai et al, 2000; Clemens et al, 1994; Clerzius et al, 2009; Clerzius et al, 2011; Clerzius et al, 2013; Daher et al, 2009; Endo-Munoz et al, 2005; Ong et al, 2005; Samuel, 1992; Sanghvi and Steel, 2011; Singh et al, 2011). Those studies principally analyzed levels of PKR-P and other proteins in post-nuclear cytoplasmic extracts from whole cell cultures.…”

Section: Resultssupporting

confidence: 87%

“…PKR is potently activated in vitro by the dimerized TAR region of gRNA (Cai et al, 2000; Daher et al, 2009; Heinicke et al, 2009; Nallagatla et al, 2011). In contrast, PKR is inhibited in extracts from HIV-1 infected or transfected cultures, at least in part by these other proteins with dsRBDs (Brand et al, 1997; Cai et al, 2000; Clerzius et al, 2009; Clerzius et al, 2011; Clerzius et al, 2013; Daher et al, 2009; Endo-Munoz et al, 2005; Ong et al, 2005; Sanghvi and Steel, 2011; Singh et al, 2011). …”

Section: Introductionmentioning

confidence: 99%

Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies

et al. 2016

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APOBEC3 cytidine deaminases and viral genomic RNA (gRNA) occur in virions, polysomes, and cytoplasmic granules, but have not been tracked together. Moreover, gRNA traffic is important, but the factors that move it into granules are unknown. Using in situ hybridization of transfected cells and protein synthesis inhibitors that drive mRNAs between locales, we observed APOBEC3F cotrafficking with gRNA without altering its movements. Whereas cells with little cytoplasmic gRNA were translationally active and accumulated Gag, suprathreshold amounts induced autophosphorylation of the cytoplasmic double-stranded RNA (dsRNA)-dependent protein kinase (PKR), causing eIF2α phosphorylation, protein synthesis suppression, and gRNA sequestration in stress granules. Additionally, we confirmed recent evidence that PKR is activated by chromosome-associated cellular dsRNAs after nuclear membranes disperse in prophase. By arresting cells in G2, HIV-1 blocks this mechanism for PKR activation and eIF2α phosphorylation. However, cytopathic membrane damage in CD4- and coreceptor-positive cultures infected with laboratory-adapted fusogenic HIV-1LAI eventually enabled PKR entry and activation in interphase nuclei. These results reveal multiple stages in the PKR-HIV-1 battleground that culminate in cell death. We discuss evidence suggesting that HIV-1s evolve in vivo to prevent or delay PKR activation by all these mechanisms.

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“…HIV-1 Tat is phosphorylated by the interferon-induced, double-stranded RNA-dependent serine/threonine protein kinase, PKR, and the cell cycle-dependent kinase CDK2/cyclin E complex. Both modifications stimulate Tat transcriptional activity (Ammosova et al, 2006; Endo-Munoz et al, 2005). However, the larger role of Tat phosphorylation in HIV-1 infection remains unclear.…”

Section: Posttranslational Modifications Regulate Tat Transcriptionalmentioning

confidence: 99%

The Control of HIV Transcription: Keeping RNA Polymerase II on Track

Ott

Geyer

Zhou

2011

Cell Host & Microbe

249

273

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SUMMARY Thirteen years ago, human cyclin T1 was identified as part of the positive transcription elongation factor b (P-TEFb) and the long-sought host cofactor for the HIV-1 transactivator Tat. Recent years have brought new insights into the intricate regulation of P-TEFb function and its relationship with Tat, revealing novel mechanisms for controlling HIV transcription and fueling new efforts to overcome the barrier of transcriptional latency in eradicating HIV. Moreover, the improved understanding of HIV and Tat forms a base for studying transcription elongation control in general. Here, we review advances in HIV transcription research with a focus on the growing family of cellular P-TEFb complexes, structural insights into the interactions between Tat, P-TEFb and TAR RNA, and the multifaceted regulation of these interactions by posttranscriptional modifications of Tat.

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Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

Cited by 56 publications

References 41 publications

p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies

The Control of HIV Transcription: Keeping RNA Polymerase II on Track

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