Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat

BACKGROUND: The nuclear transcription factor NF-κB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus (KSHV, also known as Human Herpes virus 8)-encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR. RESULTS: We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-κB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-κB pathway. K13 mutants that lacked the ability to activate the NF-κB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-κB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-κB pathway. CONCLUSIONS: Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-κB pathway which cooperates with HIV-1 Tat protein

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine

BACKGROUND: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness.To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat. RESULTS: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors. CONCLUSION: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-0

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>onstruct (75 ng/well), and the experiment was performed as described under "Materials and Methods." The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. A dose-response analysis of HIV-1 LTR activation by K13 and pro-inflammatory cytokines. 293T cells were transfected with the indicated amounts of a K13 expression plasmid and luciferase assay performed 36 h post-transfection as described for (A). The total amount of transfected DNA was kept constant by adding an empty vector. For experiments involving TNF-α and IL-1β, cells were treated with the indicated concentration of cytokines 12 h after transfection of the reporter plasmids and assayed for reporter activity after 24 h of stimulation. C. K13 activates HIV-1 LTR in Cos-7 cells. The experiment was performed as described in 1A except LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) was used for transfection and Renilla luciferase was used for normalization. D. K13 activates HIV-1 LTR in Jurkat cells. The experiment was performed as described for 1C by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA)

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-1

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>(75 ng/well) and an pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) and luciferase reporter assay performed as described in Fig. 1A. The values shown are averages (mean ± SEM) of one representative experiment out of three in which each transfection was performed in duplicate. B. HIV-1 LTR activation by wild-type and mutant K13 constructs. The experiment was performed as described for Fig. 1A

COX5B Regulates MAVS-mediated Antiviral Signaling through Interaction with ATG5 and Repressing ROS Production

<div><p>Innate antiviral immunity is the first line of the host defense system that rapidly detects invading viruses. Mitochondria function as platforms for innate antiviral signal transduction in mammals through the adaptor protein, MAVS. Excessive activation of MAVS-mediated antiviral signaling leads to dysfunction of mitochondria and cell apoptosis that likely causes the pathogenesis of autoimmunity. However, the mechanism of how MAVS is regulated at mitochondria remains unknown. Here we show that the Cytochrome c Oxidase (CcO) complex subunit COX5B physically interacts with MAVS and negatively regulates the MAVS-mediated antiviral pathway. Mechanistically, we find that while activation of MAVS leads to increased ROS production and COX5B expression, COX5B down-regulated MAVS signaling by repressing ROS production. Importantly, our study reveals that COX5B coordinates with the autophagy pathway to control MAVS aggregation, thereby balancing the antiviral signaling activity. Thus, our study provides novel insights into the link between mitochondrial electron transport system and the autophagy pathway in regulating innate antiviral immunity.</p> </div

COX5B mediates MAVS signaling by repressing ROS production.

<p>(A) HEK293 cells were pretreated with 10 ug/ml antimycin A for 2 h, then transfected with the indicated plasmids. Twenty-four hours after transfection, cells were lysed to measure the IFNβ induction. (B) HEK293 cells were pretreated with 250 or 500 µM Mito-TEMPO for 4 h, and then transfected with IFNβ reporter and pRSV/LacZ vectors together with empty vector or MAVS, or infected with VSVΔM51-GFP (MOI = 0.1). Cells were lysed to measure IFNβ activity after 24 h transfection or 10 h infection. (C and D) COX5B RNAi oligos or NC were transfected into HEK293 cells, after 20 h transfection, empty vector or MAVS plasmids were transfected again, 30 h after the second transfection, cells were collected for FACS analysis to check cellular or mitochondrial ROS production by staining with DCF (C) or MitoSOX (D), respectively. Results are presented relative to the FACS mean fluorescence intensity over control cells. (E) HEK293 cells were transfected with COX5B RNAi oligo or NC, 36 h after transfection, cells were pretreated with 250 µM Mito-TEMPO for 4 h, and transfected again with indicated plasmids. Twenty-four hours after second transfection, cells were lysed to measure the IFNβ induction. (F) After first transfection and treatment as described in (E), cells were transfected with IFNβ reporter and pRSV/LacZ vectors, followed by 16 h Sendai virus infection, cells were harvested for luciferase assays. (G) The experiments were carried out as in (F) except that cells were pretreated with 100 µM PDTC. (H) HEK293 cells were transfected with the indicated plasmids for 30 h, and then stained with MitoSOX followed by FACS analysis. Cells were treated with 0.1 µM H₂O₂ for 30 min as positive control. Results are presented relative to the FACS mean fluorescence intensity in control cells. (I) HEK293 cells were transfected with increasing amounts of MAVS expression plasmids, and empty vector was used to balance the total DNA amount. Total protein was prepared and subjected to immunoblot analysis after 24 h transfection. Data from A–B, E–G are representative of at least three independent experiments, (mean and s.d. of duplicate assays), and data from C, D and H are presented as mean ± SD from at least three independent experiments. *, P<0.05; **, P<0.01; *** P<0.001 versus control groups.</p

Overexpression of COX5B suppresses MAVS-mediated antiviral signaling.

<p>(A–C) MAVS and COX5B, or empty expression vectors were transfected in HEK293 cells together with luciferase reporter constructs driven by promoters of genes encoding IFNβ (A), NF-κB (B) or ISRE(C), as well as pRSV/LacZ as an internal control. Twenty-four hours after transfection, the luciferase activity was measured and normalized based on β-galactosidase activity. Results are presented relative to the luciferase activity in control cells. (D–F) RIG-I(N) and COX5B or empty expression vectors were transfected in HEK293 cells together with IFNβ-luc (D), NF-κB-luc (E) or IRSE-luc (F) along with pRSV/LacZ. Subsequently, cells were lysed for luciferase assays. (G) HEK293 cells were transfected with empty expression vector or COX5B for 24 h. The cells were then untreated or infected with Sendai virus (50HA units/ml) for 12 h, total RNA was isolated to check the expression of IFNβ mRNA by real-time PCR. (H) Hela cells were transfected with COX5B-GFP or COX5BΔTP-GFP and Flag-MAVS, the cells were then stained with the anti-Flag antibody and imaged by confocal microscopy. (I) HEK293 cells were transfected with the indicated constructs together with IFNβ reporter plasmids. Cells were lysed for luciferase assays after 24 h transfection. (J) HEK293 cells were transfected similarly as in (I), except that RIG-I(N) construct was used instead of MAVS. Data from A–G, I and J are representative of at least three independent experiments (mean and s.d. of duplicate or triplicate assays). *, P<0.05; **, P<0.01 versus the control groups.</p

COX5B negatively controls the virus amplification.

<p>(A–C) HEK293 cells were transfected with COX5B RNAi oligos or NC. After 48 h transfection, cells were infected by Sendai virus for 12 h, and then lysed to isolate RNA to check the transcription levels of IFNβ (A), RANTES (B) and Viperin (C) by real-time PCR. (D–F) The transfection were performed as in (A–C), except that cells were infected by VSVΔM51-GFP (MOI = 0.1) for 9 h. (G) The RNAi oligo transfection was carried out as in (A), RNAi cells were infected by Sendai virus for 16 h, and the supernatant was collected for measurement of IFNβ by ELISA. (H–J) HEK293 cells were transfected with COX5B RNAi oligos for 48 h, then cells were infected by VSV-GFP (H) or VSVΔM51-GFP (I) at the MOI of 0.01 for 12 h, subsequently the culture supernatants were collected to measure virus titer by plaque assay, or cells were imaged by fluorescence microscopy (J). Data from A–I are representative of at least three independent experiments, (A–F, mean and s.d. of triplicate assays, G–I using duplicate assays). *, P<0.05; **, P<0.01 versus the control groups.</p

Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.

Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA). Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation